Primary murine microglia are resistant to nitric oxide inhibition of indoleamine 2,3-dioxygenase

Abstract

Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that is activated by proinflammatory cytokines, including interferon-γ (IFNγ), and metabolizes tryptophan along the kynurenine pathway. Activation of murine macrophages induces not only IDO but also nitric oxide synthase (iNOS), and the ensuing production of nitric oxide (NO) inhibits IDO. To determine the sensitivity of primary cultures of murine microglia to NO, microglia were stimulated with recombinant murine IFNγ (1 ng/ml) and lipopolysaccharide (LPS) (10 ng/ml). This combination of IFNγ+LPS synergized to produce maximal amounts of nitrite as early as 16h. Steady-state mRNAs for both iNOS and IDO were significantly increased by IFNγ+LPS at 4h post-treatment, followed by an increase in IDO enzymatic activity at 24h. Murine microglia (>95% CD11b(+)) were pretreated with the iNOS inhibitor, L-NIL hydrochloride, at a dose (30 μM) that completely abrogated production of nitrite. L-NIL had no effect on IDO mRNA at 4h or IDO enzymatic activity at 24h following stimulation with IFNγ+LPS. These data establish that IDO regulation in murine microglia is not restrained by NO, thereby permitting the accumulation of kynurenine and its downstream metabolites in the central nervous system.

Figure 1

Microglia are resistant to nitric oxide inhibition of indoleamine 2, 3 dioxygenase. Primary mouse microglia were primed with IFNγ (1 ng/ml) and triggered with LPS (10 ng/ml) in the presence or absence of iNOS inhibitor L-NIL (30 μM) for 4 h and 24 h, respectively. (A) Co-stimulation with IFNγ + LPS strongly enhanced iNOS mRNA (Ct cycle for IFNγ + LPS treatment was 19 ± 0.4) compared to constitutive iNOS mRNA in primary microglia incubated in medium alone (Ct cycle was 30 ± 0.5). L-NIL had no effect on expression of iNOS transcripts (Ct cycle for IFNγ + LPS treatment with L-NIL was 19 ± 0.2). (B) IFNγ + LPS significantly increased nitrate accumulation 24 h later, and this accumulation of nitrite was blocked by L-NIL. (C) No IDO mRNA was detected in microglia incubated for 4 h in medium (>40 amplification cycles). However, IFNγ + LPS increased expression of steady-state IDO transcripts by over a 1,000-fold, and this increase in IDO mRNA was not affected by L-NIL (Ct cycles for IFNγ + LPS treatment were 29 ± 0.7 in the absence of L-NIL and 29 ± 0.3 in the presence of L-NIL). (D) IDO enzymatic activity in microglia was increased by the combination of IFNγ + LPS, and this increase was not affected by inhibition of iNOS. ** p < 0.01 compared to control medium. Each bar represents the mean ± SEM of results from 3 separate experiments.