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

Nitric oxide is an intercellular signaling molecule whose numerous fun ctions 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 in ducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-gamma and lipo polysaccharide, express inducible nitric oxide synthase. The latter wa s detected immunocytochemically in interferon-gamma-lipopolysaccharide -treated retinal pigment epithelium using rabbit antiserum to a synthe tic peptide of mouse nitric oxide synthase. Untreated cultures of reti nal pigment epithelium or cultures treated with either interferon-gamm a or lipopolysaccharide alone were not immunoreactive. Induction of iN OS in gamma-interferon-lipopolysaccharide-stimulated retinal pigment e pithelial cells was also evidenced by the presence of nitric oxide syn thase enzyme activity in lysates of stimulated but net unstimulated re tinal pigment epithelial cells. On immunoblots of lysates of stimulate d murine retinal pigment epithelial cells, rabbit antiserum to iNOS re cognized 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 cel ls treated with only interferon-gamma or lipopolysaccharide alone. Nit rite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-gamm a and lipopolysaccharide, and continued to accumulate in a linear fash ion for at least 96 hr. Treatment of cultured retinal pigment epitheli um with interferon-gamma, lipopolysaccharide and either basic fibrobla st growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensifie d signals on immunoblots, enhanced accumulation of nitrite and increas ed iNOS enzyme activity. Conversely, when transforming growth factor-b eta was present in the culture medium, gamma-interferon-LPS-induced ex pression of nitric oxide synthase and NO release were reduced. We conc lude that interferon-gamma synergizes with lipopolysaccharide to induc e synthesis of inducible nitric oxide synthase and production of nitri c oxide by murine retinal pigment epithelium and that this induction c an be modulated by basic fibroblast growth factor, epidermal growth fa ctor or transforming growth factor-beta.