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(Investigative Ophthalmology and Visual Science. 1999;40:1899-1905.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Differential Expression of Nitric Oxide Synthase in Experimental Uveoretinitis

Jie Zhang12, Lan-Ying Wu3, Guey-Shuang Wu12 and Narsing A. Rao12

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){gamma}/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{gamma}/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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