CYTOKINE REGULATION OF NITRIC-OXIDE SYNTHASE IN MOUSE RETINAL-PIGMENT EPITHELIAL-CELLS IN CULTURE

Nitric oxide is an intercellular signaling molecule whose numerous functions include regulation of vascular tone, mediation of the cytotoxic effects of macrophages and potentiation of synaptic transmission. For some cellular functions, nitric oxide synthesis is mediated by the inducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-gamma and lipopolysaccharide, express inducible nitric oxide synthase. The latter was detected immunocytochemically in interferon-gamma-lipopolysaccharide-treated retinal pigment epithelium using rabbit antiserum to a synthetic peptide of mouse nitric oxide synthase. Untreated cultures of retinal pigment epithelium or cultures treated with either interferon-gamma or lipopolysaccharide alone were not immunoreactive. Induction of iNOS in gamma-interferon-lipopolysaccharide-stimulated retinal pigment epithelial cells was also evidenced by the presence of nitric oxide synthase enzyme activity in lysates of stimulated but net unstimulated retinal pigment epithelial cells. On immunoblots of lysates of stimulated murine retinal pigment epithelial cells, rabbit antiserum to iNOS recognized a 130-kDa protein which comigrated with the inducible nitric oxide synthase of macrophages and which was not detectable in lysates of unstimulated retinal pigment epithelial cells nor in lysates of cells treated with only interferon-gamma or lipopolysaccharide alone. Nitrite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-gamma and lipopolysaccharide, and continued to accumulate in a linear fashion for at least 96 hr. Treatment of cultured retinal pigment epithelium with interferon-gamma, lipopolysaccharide and either basic fibroblast growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensified signals on immunoblots, enhanced accumulation of nitrite and increased iNOS enzyme activity. Conversely, when transforming growth factor-beta was present in the culture medium, gamma-interferon-LPS-induced expression of nitric oxide synthase and NO release were reduced. We conclude that interferon-gamma synergizes with lipopolysaccharide to induce synthesis of inducible nitric oxide synthase and production of nitric oxide by murine retinal pigment epithelium and that this induction can be modulated by basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta.