title: Differential expression of nitric oxide synthase in experimental uveoretinitis.
abstract: OBJECTIVE
  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 IFNgamma/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.
authors.affiliation: Doheny Eye Institute, Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles 90033-1088, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: L Y
authors.initials: LY
authors.lastname: Wu
authors.forename: G S
authors.initials: GS
authors.lastname: Wu
authors.forename: N A
authors.initials: NA
authors.lastname: Rao
mesh_terms: D000818:Animals; D019393:Arrestin / Q000494:pharmacology; D015152:Blotting, Northern; D002417:Cattle; D002478:Cells, Cultured; D005136:Eye Proteins; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D004676:Myelin Basic Protein / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011917:Rats, Inbred Lew; D012173:Retinitis / Q000139:chemically induced / Q000201:enzymology* / Q000473:pathology; D012177:Retinol-Binding Proteins / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014605:Uveitis / Q000139:chemically induced / Q000201:enzymology* / Q000473:pathology; D000099214:Retinol-Binding Proteins, Interstitial
chemical_list: D019393:Arrestin; D005136:Eye Proteins; D004676:Myelin Basic Protein; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger; D012177:Retinol-Binding Proteins; D012710:Serum Albumin, Bovine; D000099214:Retinol-Binding Proteins, Interstitial; C496319:NOS2 protein, human; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat