Ascorbate in Aqueous Humor Augments Nitric Oxide Production by Macrophages

Abstract

Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting pro-inflammatory nitric oxide (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 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, CAT2B, and increased L-arg uptake in Mϕs stimulated in the presence of AqH. Arginases were expressed by stimulated Mϕs but competition for L-arg with NOS2 was excluded. Expression of GTP cyclohydrolase which produces tetrahydrobiopterin (H4B), an essential cofactor for NOS2 homodimerization, increased after Mϕ 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 H4B. Therefore, AqH may augment NO production in macrophages by stabilizing H4B and increasing intracellular L-Arg.

Does vitamin C enhance nitric oxide bioavailability in a tetrahydrobiopterin-dependent manner? In vitro, in vivo and clinical studies

“…Another study did not observe any changes in iNOS expression in mouse macrophage RAW 264.7 cells incubated with Asc, in contrast to a previous study that found a 2-fold increase in iNOS levels in mouse macrophage J774.1 cells [41], but did find an increase in BH 4 level and NOS activity [38]. Likewise, Asc in aqueous humor was found to increase nitrite formation in mice macrophages, an effect that was ascribed to a stabilizing effect on BH 4 observed in the growth medium [42]. In polymorphonuclear leukocytes, both Asc and DHA were found to increase NOS activity [34].…”

Does vitamin C enhance nitric oxide bioavailability in a tetrahydrobiopterin-dependent manner? In vitro, in vivo and clinical studies

“…Another study did not observe any changes in iNOS expression in mouse macrophage RAW 264.7 cells incubated with Asc, in contrast to a previous study that found a 2-fold increase in iNOS levels in mouse macrophage J774.1 cells [41], but did find an increase in BH 4 level and NOS activity [38]. Likewise, Asc in aqueous humor was found to increase nitrite formation in mice macrophages, an effect that was ascribed to a stabilizing effect on BH 4 observed in the growth medium [42]. In polymorphonuclear leukocytes, both Asc and DHA were found to increase NOS activity [34].…”

Splenectomy Promotes Indirect Elimination of Intraocular Tumors by CD8+ T Cells That Is Associated with IFNγ- and Fas/FasL-Dependent Activation of Intratumoral Macrophages

“…NO is synthesized in activated Mϕs by the enzyme NO synthase 2 (NOS2) (17). Therefore, to determine whether a similar mechanism was involved in rejection of intraocular tumors in SPLNX mice, we compared the frequency of NOS2 + Mϕs by flow cytometry in day 16 Luc-EG7 intraocular tumors of SPLNX and control B6, gld, lpr and IFNγR1 −/− mice.…”

Deletion of Vitamin C biosynthesis enzyme, Arabino‐1, 4‐lactone oxidase in Leishmania donovani results in increased pro‐inflammatory responses from host immune cells

“…Growth rate experiments were conducted by inoculating stationary‐phase parasites at a density of 1 × 10 6 cells ml −1 in standard M199 medium with 5% FBS in 25 cm 2 flasks without respective selection drug and culturing at 22°C. Growth of the parasites was also examined in M199 media (5% FBS) pre‐treated with ascorbate oxidase (Sigma‐Aldrich) (10 U ml −1 ) for 1 h at room temperature as previously described (McKenna et al ., ). Growth rates of each of the cultures were determined at 24 h intervals by using a Neubauer haemocytometer.…”

Does vitamin C enhance nitric oxide bioavailability in a tetrahydrobiopterin-dependent manner? In vitro, in vivo and clinical studies

“…Another study did not observe any changes in iNOS expression in mouse macrophage RAW 264.7 cells incubated with Asc, in contrast to a previous study that found a 2-fold increase in iNOS levels in mouse macrophage J774.1 cells [41], but did find an increase in BH 4 level and NOS activity [38]. Likewise, Asc in aqueous humor was found to increase nitrite formation in mice macrophages, an effect that was ascribed to a stabilizing effect on BH 4 observed in the growth medium [42]. In polymorphonuclear leukocytes, both Asc and DHA were found to increase NOS activity [34].…”

Splenectomy Promotes Indirect Elimination of Intraocular Tumors by CD8+ T Cells That Is Associated with IFNγ- and Fas/FasL-Dependent Activation of Intratumoral Macrophages

“…NO is synthesized in activated Mϕs by the enzyme NO synthase 2 (NOS2) (17). Therefore, to determine whether a similar mechanism was involved in rejection of intraocular tumors in SPLNX mice, we compared the frequency of NOS2 + Mϕs by flow cytometry in day 16 Luc-EG7 intraocular tumors of SPLNX and control B6, gld, lpr and IFNγR1 −/− mice.…”

Deletion of Vitamin C biosynthesis enzyme, Arabino‐1, 4‐lactone oxidase in Leishmania donovani results in increased pro‐inflammatory responses from host immune cells

“…Growth rate experiments were conducted by inoculating stationary‐phase parasites at a density of 1 × 10 6 cells ml −1 in standard M199 medium with 5% FBS in 25 cm 2 flasks without respective selection drug and culturing at 22°C. Growth of the parasites was also examined in M199 media (5% FBS) pre‐treated with ascorbate oxidase (Sigma‐Aldrich) (10 U ml −1 ) for 1 h at room temperature as previously described (McKenna et al ., ). Growth rates of each of the cultures were determined at 24 h intervals by using a Neubauer haemocytometer.…”

Does vitamin C enhance nitric oxide bioavailability in a tetrahydrobiopterin-dependent manner? In vitro, in vivo and clinical studies

“…Another study did not observe any changes in iNOS expression in mouse macrophage RAW 264.7 cells incubated with Asc, in contrast to a previous study that found a 2-fold increase in iNOS levels in mouse macrophage J774.1 cells [41], but did find an increase in BH 4 level and NOS activity [38]. Likewise, Asc in aqueous humor was found to increase nitrite formation in mice macrophages, an effect that was ascribed to a stabilizing effect on BH 4 observed in the growth medium [42]. In polymorphonuclear leukocytes, both Asc and DHA were found to increase NOS activity [34].…”

Splenectomy Promotes Indirect Elimination of Intraocular Tumors by CD8+ T Cells That Is Associated with IFNγ- and Fas/FasL-Dependent Activation of Intratumoral Macrophages

“…NO is synthesized in activated Mϕs by the enzyme NO synthase 2 (NOS2) (17). Therefore, to determine whether a similar mechanism was involved in rejection of intraocular tumors in SPLNX mice, we compared the frequency of NOS2 + Mϕs by flow cytometry in day 16 Luc-EG7 intraocular tumors of SPLNX and control B6, gld, lpr and IFNγR1 −/− mice.…”

Deletion of Vitamin C biosynthesis enzyme, Arabino‐1, 4‐lactone oxidase in Leishmania donovani results in increased pro‐inflammatory responses from host immune cells

“…Growth rate experiments were conducted by inoculating stationary‐phase parasites at a density of 1 × 10 6 cells ml −1 in standard M199 medium with 5% FBS in 25 cm 2 flasks without respective selection drug and culturing at 22°C. Growth of the parasites was also examined in M199 media (5% FBS) pre‐treated with ascorbate oxidase (Sigma‐Aldrich) (10 U ml −1 ) for 1 h at room temperature as previously described (McKenna et al ., ). Growth rates of each of the cultures were determined at 24 h intervals by using a Neubauer haemocytometer.…”