Differential expression of nitric oxide synthase in experimental uveoretinitis

Jie Zhang, Lan Ying Wu, Guey Shuang Wu, Narsing A. Rao

Research output: Contribution to journalArticle

30 Citations (Scopus)

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)γ/lipopolysaccharide (LPS) or S-antigen and to interphoto-receptor 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. Ill contrast, a large number of EDI+ and OX6+ cells that were localized in the urea 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.

Original languageEnglish (US)
Pages (from-to)1899-1905
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume40
Issue number9
StatePublished - Aug 1 1999
Externally publishedYes

Fingerprint

Nitric Oxide Synthase Type II
Nitric Oxide Synthase
Macrophages
Retina
Retinol-Binding Proteins
Antigens
Interferons
Lipopolysaccharides
Rabbits
Peptides
Antigen Receptors
Proteins
Rhodamines
Retinal Pigments
Myelin Basic Protein
Streptavidin
Antibodies
Conjunctiva
Glial Fibrillary Acidic Protein
Frozen Sections

All Science Journal Classification (ASJC) codes

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Differential expression of nitric oxide synthase in experimental uveoretinitis. / Zhang, Jie; Wu, Lan Ying; Wu, Guey Shuang; Rao, Narsing A.

In: Investigative Ophthalmology and Visual Science, Vol. 40, No. 9, 01.08.1999, p. 1899-1905.

Research output: Contribution to journalArticle

Zhang, Jie ; Wu, Lan Ying ; Wu, Guey Shuang ; Rao, Narsing A. / Differential expression of nitric oxide synthase in experimental uveoretinitis. In: Investigative Ophthalmology and Visual Science. 1999 ; Vol. 40, No. 9. pp. 1899-1905.
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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)γ/lipopolysaccharide (LPS) or S-antigen and to interphoto-receptor 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. Ill contrast, a large number of EDI+ and OX6+ cells that were localized in the urea 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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N2 - 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 interphoto-receptor 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. Ill contrast, a large number of EDI+ and OX6+ cells that were localized in the urea 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.

AB - 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 interphoto-receptor 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. Ill contrast, a large number of EDI+ and OX6+ cells that were localized in the urea 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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