Ascorbate in aqueous humor augments nitric oxide production by macrophages

Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting proinflammatory NO production by macrophages (MΦs). Consistent with previous observations, we observed that although MΦs stimulated in the presence of AqH expressed NO synthase 2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure of NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of L-arginine (L-Arg) into L-citrulline, was augmented in lysates of MΦs stimulated in the presence of AqH. These data suggested that intracellular L-Arg may have been limited by AqH. However, we observed increased mRNA expression of the L-Arg transporter, cationic amino acid transporter 2B, and increased L-Arg uptake in MΦs stimulated in the presence of AqH. Arginases were expressed by stimulated Ms, but competition for L-Arg with NOS2 was excluded. Expression of GTP cyclohydrolase, which produces tetrahydrobiopterin (H(4)B), an essential cofactor for NOS2 homodimerization, increased after M stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together, these data provided no evidence for inhibited NOS2 enzymatic activity by AqH, suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH, and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in MΦs via ascorbate, which limited degradation of H(4)B. Therefore, AqH may augment NO production in macrophages by stabilizing H(4)B and increasing intracellular L-Arg.