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1 From the Doheny Eye Institute, the 2 Department of Ophthalmology, and 3 Kenneth R. Norris Jr. Cancer Hospital and Research Institute, University of Southern California, School of Medicine, Los Angeles.
Abstract
PURPOSE. To investigate the site and the cellular source of inducible nitric oxide synthase (iNOS) expression in human S-antigen peptide–induced experimental autoimmune uveoretinitis (EAU).
METHODS. Twenty-one Lewis rats were sensitized with human S-antigen peptides.
Three rats were killed each consecutive day from day 6 through day 12
after sensitization. Frozen sections of the enucleated eyes were
analyzed for iNOS by the dual immunohistochemical method. Primary
antibodies included rabbit anti-mouse iNOS combined with anti-human
endothelium NOS, anti-rat lysosomal protein (ED1), or anti-rat major
histocompatibility complex class II molecule (OX6) monoclonal
antibodies. Secondary antibodies were fluorescein-conjugated anti-mouse
IgG and streptavidin rhodamine-labeled anti-rabbit IgG. The adjacent
sections were separately stained with ED1, iNOS, and glial fibrillary
acidic protein (GFAP). The mouse macrophage cell line RAW 264.7 was
exposed to either interferon (IFN)
/lipopolysaccharide (LPS) or
S-antigen and to interphotoreceptor retinoid-binding protein (IRBP),
myelin basic protein, and bovine serum albumin for 12 hours. Cells were
harvested for detection of iNOS expression by northern blot analysis
hybridization and detection of protein by immunohistochemistry.
RESULTS. In the retina of eyes with EAU, ED1+/iNOS+ and OX6+/iNOS+ cells were
first detected on day 9 after sensitization. These iNOS+ cells
increased in number on subsequent days in parallel with the increasing
severity of retinal damage. Most of the cells localized around the
outer retina. In contrast, a large number of ED1+ and OX6+ cells that
were localized in the uvea and conjunctiva were negative for iNOS.
Retinal pigment epithelial cells did not stain for iNOS. Macrophages
exposed to IFN/LPS, S-antigen, and IRBP showed expression of iNOS
mRNA and the protein.
CONCLUSIONS. Macrophages are an important source of NO production in eyes with EAU. These macrophages preferentially express iNOS in the retina. Such a differential expression of iNOS by the macrophages appears to be related to retinal soluble proteins.
Nitric oxide (NO) has been widely studied by those attempting to elucidate the mechanism of inflammation and tissue destruction.1 The cytotoxic molecule NO is synthesized by an inducible NO synthase (iNOS); once this synthase is expressed, NO is produced for a period of several hours to several days. In contrast, constitutive NO synthase (cNOS) generates NO for short periods of only a few seconds to a few minutes. Cytotoxicity is usually associated with the products of iNOS rather than the products of cNOS.1 Increased production of NO is found in many endotoxin-induced acute inflammations, including endotoxin-induced uveitis (EIU).2 3 4 The release of NO has also been noted in various experimental autoimmune inflammatory disorders, such as experimental arthritis and certain neurologic inflammations.5 6 Such experimentally induced inflammations have shown that NO production is involved in the enhancement of inflammation through its effects on vasodilatation, neutrophil adhesion, and alteration of vascular permeability leading to tissue damage.
Unlike EIU, experimental autoimmune uveoretinitis (EAU) can be readily induced by several retinal proteins, such as retinal soluble antigen (S-antigen), interphotoreceptor retinoid-binding protein (IRBP), rhodopsin, and others. Experimental autoimmune uveoretinitis is clinically and histologically different from EIU. In the latter, the inflammatory process is mild and involves primarily the anterior uvea. In contrast, the inflammation of EAU is characterized by retinitis, marked retinal damage, and uveitis. The retinal disease is mainly localized in the outer retina, and the ensuing retinal damage could be caused by the formation of oxygen metabolites, including peroxynitrite.7 8 In the present study, we attempted to investigate the cellular source of and the role of retinal proteins in the expression of iNOS and NO generation that occurs with S-antigen peptide–induced uveoretinitis.
Methods
Twenty-one Lewis rats (aged 6–8 weeks) were immunized by a hind footpad injection of 0.1 ml synthetic human S-antigen peptide (100 µg) in Freund’s complete adjuvant containing 4 mg/ml of heat-killed Mycobacterium tuberculosis. Three rats were killed on each day, from day 6 to day 12 after immunization. Three naive rats served as control subjects. All animal procedures used in this study were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Antigen Preparation
Human S-Antigen Peptide.
The sequence of human S-antigen peptide, 307 to 326 DTNLASSTIIKEGI
DRTVLG,9
was synthesized by an automated peptide
synthesizer (model 430A; Applied Biosystems, Foster City, CA), desalted
on a Sephadex G-10 column (Sigma, St. Louis, MO), and purified by
reversed-phase high-performance liquid chromatography (RP 304 Column;
Bio-Rad Laboratories, Hercules, CA). This peptide is highly identical
with the amino acid sequence of bovine S-antigen and is uveitogenic for
Lewis rats.10
Bovine S-Antigen and IRBP.
Bovine S-antigen and IRBP were isolated according to a procedure
previously described.7
11
Briefly, each antigen was
precipitated from bovine retinal extract by half-saturated ammonium
sulfate, then purified by gel filtration chromatography (Ultrogel AcA
34 followed by HAultrogel; IBF Biotechnics, Savage, MD). Both antigens
were identified in elutes by the Ouchterlony immunodiffusion
test (Biowhittaker, Walkersville, MD). On 10% sodium dodecyl sulfate
(SDS)–polyacrylamide gel electrophoresis, isolated bovine S-antigen or
IRBP revealed one band of approximately 48 kDa in S-antigen and one of
approximately 130 kDa in IRBP. The uveitogeneity was separately
confirmed by immunization of Lewis rats.
Immunolocalization of iNOS in the Ocular Tissues
The enucleated eyes were embedded in frozen tissue specimen
medium, and 7-µm frozen sections were obtained from the
globes. These sections were fixed in acetone and exposed to various
antibodies. The primary antibodies used included rabbit anti-mouse
macrophage iNOS polyclonal antibody (1:50) and mouse anti-human
endothelium NOS monoclonal antibody (cNOS, 1:50), both from
Transduction Laboratories (Lexington, KY), and mouse anti-rat lysosomal
membrane (ED1, 1:100) and mouse anti-rat major histocompatibility
complex class II common determinant (OX6, 1:100) monoclonal antibodies
(Serotec). Each section was incubated with one of the following
combinations of primary antibodies: iNOS/cNOS, iNOS/ED1, and iNOS/OX6
at 4°C overnight. After a phosphate-buffered saline (PBS) wash, the
sections were incubated with two different secondary antibodies, goat
anti-mouse fluorescein-conjugated IgG (1:200; Vector Laboratories,
Burlingame, CA) and goat anti-rabbit streptavidin rhodamine-labeled IgG
(1:100; Southern Biotech, Birmingham, AL), consecutively at room
temperature for 2 hours. After a PBS wash, the specimens were mounted
and examined under a confocal laser scanning microscope (Carl Zeiss,
Oberkochen, Germany). For control, mouse IgG (20 µg/ml) and normal
rabbit serum (1:750) were substituted for the primary antibodies. The
adjacent sections of each eye were stained with hematoxylin and eosin.
The adjacent sections were separately incubated with antibodies against ED1, iNOS, and rabbit anti-cow glial fibrillary acidic protein serum (GFAP, 1:100, Dako, Carpinteria, CA). The following secondary antibodies were used: biotin-conjugated anti-rabbit immunoglobulins for iNOS (1:300, Dako), anti-mouse immunoglobulins for ED1 (1:100, Dako, preabsorbed with normal rat serum), and goat anti-rabbit streptavidin rhodamine-labeled IgG (1:100, Southern Biotech) for GFAP. The antigen–antibody binding was detected by avidin-biotinylated horseradish peroxidase (Vector Laboratories), then with 3-amino-9-ethyl-carbazole (Sigma). The sections were briefly immersed in hematoxylin for counterstaining and observed under light microscope.
Cell Culture and Induction
Mouse macrophage cell line RAW264.7 (American Type Culture
Collection, Rockville, MD) was used in vitro to detect the expression
of iNOS in the presence of retinal proteins (S-antigen and IRBP). The
cells were plated on a six-well culture plate (Becton Dickinson,
Lincoln Park, NJ) with a density of 106
cells/well. Culture media consisted of minimum essential medium (MEM,
Gibco, Grand Island, NY) containing 10% fetal bovine serum (Atlanta
Biologicals, Norcross, GA), 100 U/ml penicillin and 100 µg/ml
streptomycin sulfate (Gibco), and 2 mM L-glutamine (JRH
Biosciences, Lenexa, KS).
On reaching confluence, the macrophage cultures were exposed to media
containing one of the following agents (50 µg/ml of each): bovine
retinal S-antigen, IRBP, bovine serum albumin (BSA; Sigma); bovine
brain myelin basic protein (MBP; Sigma) and a mixture of
lipopolysaccharide (LPS; 100 ng/ml, Sigma) with recombinant murine
IFN (100 U/ml; Gibco). The culture media, retinal proteins, BSA, and
MBP were passed through endotoxin-removing gel (Detoxi-Gel; Pierce,
Rockford, IL). All were incubated at 37°C with 10%
CO2 in an air atmosphere for 12 hours. For
negative control samples, cells were incubated without any of the
reagents mentioned.
Detection of iNOS mRNA Expression in RAW 264.7 Cell Line
The remaining macrophages were collected for isolation of total
RNA. A total RNA isolation kit (CLONsep; Clontech, Palo Alto, CA)
protocol was followed. Total RNA (10 µg per lane) was subjected to
electrophoresis on a 1% agarose gel containing 18% formaldehyde. RNAs
were blot transferred to nylon membrane (Amersham Life Science,
Arlington Heights, IL) and UV autocrosslinked. Membrane was hybridized
with iNOS cDNA probe (Cayman Chemical, Ann Arbor, MI) for 20 hours at
45°C. The hybridization buffer contained 50% deionized formamide,
4x SSC, 1x Denhardt’s, 0.05% SDS, and denatured salmon sperm DNA
(100 µg/ml, Sigma). The cDNA probe (106 cpm/ml)
was labeled with [
-32P] dCTP (Amersham Life
Science) by random priming. The hybridized membrane was washed at room
temperature in 2x SSC/0.1% SDS, followed by 1x SSC/0.1% SDS, and
then by 0.1x SSC/0.1% SDS at 65°C for 30 minutes each.
Autoradiography was performed by exposure to x-ray film (X-Omat;
Eastman Kodak, Rochester, NY) at -70°C for 12 hours in the presence
of intensifying screens. Relative mRNA levels were revealed by
subsequent stripping and rehybridized with
[-32P] dCTP-labeled
glyceraldehyde-3-phosphate dehydrogenase cDNA probe.
Detection of iNOS Expression in RAW 264.7 Cell Line
The RAW 264.7 macrophages were plated on two-well chamber slides
at a density of 102 cells/well. These cells were
incubated with S-antigen, IRBP, or BSA, at a dose of 100 µg/ml each,
or with IFN/LPS (100 U/100 ng/ml) at 37°C with 10%
CO2 for 12 hours. The slides were fixed by 100%
acetone at 4°C for 5 minutes and submitted for immunohistochemistry
using iNOS polyclonal antibody, as described earlier.
Results
Histopathologic analysis of the hematoxylin and eosin preparations revealed the presence of retinal perivasculitis, infiltrating cells in the anterior chamber and iris and ciliary body on postsensitization day 9. The inflammation peaked on day 12, and the inflammatory infiltration was present in the inner and outer retina, which consisted of monocytes, neutrophils, and lymphocytes. In addition, the infiltration was present in the subretinal space, iris, ciliary body, choroid, conjunctiva, and limbus. Retinal damage included focal edema and exudation in the inner and outer nuclear layers and destruction of photoreceptor cells. Exudative retinal detachment also was noted. The severity of EAU at various intervals is shown in Table 1 .
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/LPS, S-antigen, and
IRBP, but not in the cells exposed to BSA or in the unexposed control
cells (Fig. 5)
. Similarly, expression of iNOS mRNA was found in the macrophages
exposed to IFN/LPS, S-antigen, and IRBP, but not in the presence of
BSA or in the unexposed control cells (Fig. 6).
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In the present study, the expression of iNOS was seen in the extravasated mononuclear cells that expressed phenotypic markers for macrophages (ED1 and OX6). These cells were primarily present in the retina, particularly at the site of photoreceptor cell damage. Similarly, a colocalization of iNOS and ED1+ macrophages has been shown in the retina of animals with EAU, particularly in the rod outer segments.12 In addition, we found that iNOS staining was predominant in infiltrates of the retina rather than those in the uvea, limbus, and conjunctiva, although the infiltrating cells, positive for class II molecules (OX6) and ED1, were seen in those sites. Such findings suggest that mononuclear phagocyte extravasation and display of class II molecules may not be sufficient for iNOS expression. Exposure to the local components, such as S-antigen, IRBP, or other retinal proteins may be required for the macrophages to express iNOS in EAU.
Endotoxin and several cytokines are known to enhance in vitro
expression of iNOS. In vivo, generation of NO has been demonstrated in
EIU.2
3
4
Based on such in vivo and in vitro studies, it
appears that endotoxin mediates expression of iNOS through induction of
various cytokines. However, which cytokine or cytokines could enhance
iNOS production in EIU is not known. It is known that activated
macrophages release tumor necrosis factor- and interleukin-1 in the
presence of endotoxin and that these cytokines may be inducers of iNOS
expression.13
However, in EAU, expression of iNOS in the
class II molecule–positive macrophages was primarily seen in the
retina, even though the uveal tract was similarly infiltrated by the
macrophages. Such a differential expression may be caused by higher
levels of cytokine released in the retina compared with that in the
uveal tract. A previous report indicated that mRNA expression of IFN
and iNOS was increased in the whole eyeball in EAU.12
The preferential expression of iNOS-positive macrophages in the retina also suggests either that stimulatory factors of iNOS exist in the retina or that there are inhibitory factors of iNOS in the uvea and in the limbal conjunctiva. TGF-ß has been found to suppress NO release by macrophages and other cells.14 15 16 17 18 In vivo, TGF-ß is widely distributed in the retina, particularly in the photoreceptor layer, rather than in the iris and ciliary body.19 20 Such a distribution pattern is expected to suppress the expression of iNOS in the retina, whereas the present results disclosed a preferential expression of iNOS in the retina and a scarcity of expression in the uvea and the conjunctiva. Such data suggest that TGF-ß may not play a critical role in the modulation of iNOS expression in the macrophages infiltrating the retina in EAU.
The differential expression of iNOS in the inflammatory phagocytes in the retina compared with the uvea suggests that local factors are required for NO generation by the macrophages. The present in vitro studies reveal that soluble proteins such as S-antigen and IRBP could be local factors in the retina upregulating iNOS expression and NO release. It is plausible that other soluble proteins also may induce iNOS expression in the macrophages. Absence of such proteins in the uvea, limbus, and conjunctiva may explain the scarcity of iNOS-expressing macrophages in these inflamed tissues, despite the expression of class II molecules by such cells. Similar to the present results, thyroid autoantigens were recently found to induce expression of iNOS in monocytes isolated from patients with Graves’ disease.21 However, whether the retinal proteins can directly, or through cytokines, increase expression of iNOS is not clear and needs further investigation.
Coexpression of cNOS and iNOS was noted in the retinal microvascular endothelia, whereas endothelial cells in large vessels expressed cNOS only. In the early stages of EAU development, the infiltrating cells around the vessels were iNOS-/ED1+. As the inflammation progressed, the migrated ED1+ macrophages expressed iNOS, particularly at the outer retina, where S-antigen and IRBP are primarily localized. These findings also suggest the role of these autoantigens in the induction of iNOS within the activated macrophages.
The present study shows that iNOS and its mRNA can be induced in
the RAW 264.7 cell line when exposed to S-antigen and IRBP. We also
found that iNOS mRNA was not induced in these cells when exposed to
MBP, one of the main proteins consisting of myelin and a fairly small
molecule (14 kDa) compared with S-antigen (48 kDa) and IRBP (130 kDa).
A recent study has shown that phagocytosis of myelin, a
receptor-mediated process, by rat peritoneal macrophages is poorer than
that by microglia, but it can be increased in the presence of IFN or
opsonized myelin. Such a phagocytic process could generate NO and could
be inhibited by TGFß.22
It is conceivable that cytokines
could enhance phagocytosis of MBP by microglia and macrophages in EAU
and lead to release of NO.22
Compared with this study our
negative data may be because of our use of the purified MBP rather than
myelin extract and because there was no exposure to opsonized MBP in
the cells we used. These factors could have resulted in the absence of
phagocytosis of MBP by macrophages and subsequent nitrite production.
In conclusion, the present study shows that in EAU, macrophages are an important source of NO production. iNOS was preferentially expressed in the macrophages infiltrating the retina but scarcely in the uvea. The retina-specific proteins were found to stimulate iNOS synthesis in the macrophages. Such findings suggest that in vivo expression of iNOS in the macrophages may require local signals (i.e., activation followed by exposure to cytokines and/or autoantigens).
Footnotes
Reprint requests: Narsing A. Rao MD, Doheny Eye Institute, 1450 San Pablo Street, Los Angeles, CA 90033-1088.
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1998 (Abstract nr 3597).
Supported in part by Grants EY03040 and EY12363 from the National Institutes of Health and by an unrestricted grant from Research to Prevent Blindness.
Submitted for publication June 8, 1998; revised March 19, 1999; accepted April 9, 1999.
Proprietary interest category: N.
References
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-dependent mechanism J Immunol 159,5132-5142[Abstract]
-induction of nitric oxide synthase in rat renal mesangial cells Biochem Biophys Res Commun 175,372-379[Medline][Order article via Infotrieve]
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