Regulation of nitric oxide synthase 2 in rabbit corneal cells

Purpose. The purpose of these studies was to investigate the role of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and transforming growth factor-β (TGF-β) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells. Methods. Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR. Results. NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-γ, little or no nitrite accumulation was induced by TNF-α, IL-1β, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-γ were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-γ receptor homolog, M-T7, rRaIFN-γ, in combination with IL-1β and TNF-α, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 μM) until after 24 hours postinduction, NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-β₁ and β₂ inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor. Conclusions. In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-γ in combination with IL-1β and TNF-α but not by any of these cytokines alone, while TGF-β downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.