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Jie Zhang, Lan-Ying Wu, Guey-Shuang Wu, Narsing A. Rao; Differential Expression of Nitric Oxide Synthase in Experimental Uveoretinitis. Invest. Ophthalmol. Vis. Sci. 1999;40(9):1899-1905.
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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)γ/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 IFNγ/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.
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