Substantial increases in the tryptophan-kynurenine pathway metabolites, l-kynurenine and the neurotoxin quinolinic acid, occur in human brain, blood and systemic tissues during immune activation. Studies in vitro have shown that not all human cells are capable of synthesizing quinolinate. To investigate further the mechanisms that limit l-kynurenine and quinolinate production, the activities of kynurenine pathway enzymes and the ability of different human cells to convert pathway intermediates into quinolinate were compared. Stimulation with interferon gamma substantially increased indoleamine 2,3-dioxygenase activity and L-kynurenine production in primary peripheral blood macrophages and fetal brains (astrocytes and neurons), as well as cell lines derived from macrophage/monocytes (THP-1), U373MG astrocytoma, SKHEP1 liver and lung (MRC-9). High activities of kynurenine 3-hydroxylase, kynureninase or 3-hydroxyanthranilate 3,4-dioxygenase were found in interferon-gamma-stimulated macrophages, THP-1 cells and SKHEP1 cells, and these cells made large amounts of quinolinate when supplied with L-tryptophan, L-kynurenine, 3-hydroxykynurenine or 3-hydroxyanthranilate. Quinolinate production by human fetal brain cultures and U373MG cells was restricted by the low activities of kynurenine 3-hydroxylase, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase, and only small amounts of quinolinate were synthesized when cultures were supplied with L-tryptophan or 3-hydroxyanthranilate. In MRC-9 cells, quinolinate was produced only from 3-hydroxykynurenine and 3-hydroxyanthranilate, consistent with their low kynurenine 3-hydroxylase activity. The results are consistent with the notion that indoleamine 2,3-dioxygenase is an important regulatory enzyme in the production of L-kynurenine and quinolinate. Kynurenine 3-hydroxylase and, in some cells, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase are important determinants of whether a cell can make quinolinate.

Quinolinic acid is an excitotoxic kynurenine pathway metabolite, the concentration of which increases in human brain during immune activation. The present study compared quinolinate responses to systemic and brain immune activation in gerbils and rats. Global cerebral ischemia in gerbils, but not rats, increased hippocampus indoleamine-2,3-dioxygenase activity and quinolinate levels 4 days postinjury. In a rat focal ischemia model, small increases in quinolinate concentrations occurred in infarcted regions on days 1, 3, and 7, although concentrations remained below serum values. In gerbils, systemic immune activation by an intraperitoneal injection of endotoxin (1 mg/kg of body weight) increased quinolinate levels in brain, blood, lung, liver, and spleen, with proportional increases in lung indoleamine-2,3-dioxygenase activity at 24 h postinjection. In rats, however, no significant quinolinate content changes occurred, whereas lung indoleamine-2‚3-dioxygenase activity increased slightly. Gerbil, but not rat, brain microglia and peritoneal monocytes produced large quantities of[] tryptophan. Gerbil astrocytes produced relatively small quantities of quinolinate, whereas rat astrocytes produced no detectable amounts. These results demonstrate that the limited capacity of rats to replicate elevations in brain and blood quinolinic acid levels in response to immune activation is attributable to blunted increases in local indoleamine-2,3-dioxygenase activity and a low capacity of microglia, astrocytes, and macrophages to convert L-tryptophan to quinolinate.

IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

The metabolism of l‐tryptophan to the neuroactive kynurenine pathway metabolites, l‐kynurenine, kynurenate and quinolinate, and the effects of two inhibitors of quinolinate synthesis (6‐chlorotryptophan and 4‐chloro‐3‐hydroxyanthranilate) were investigated by mass spectrometric assays in cultured cells and in vivo. Cell lines obtained from astrocytoma, neuroblastoma, macrophage/monocytes, lung, and liver metabolized l‐[13C6]‐tryptophan to l‐[13C6]kynurenine and [13C6]kynurenate, particularly after indoleamine‐2,3‐dioxygenase induction by interferon‐γ. Kynurenine aminotransferase activity was measurable in all cell types examined but was unaffected by interferon‐γ. These results suggest that many cell types can be sources of kynurenate following immune activation. In vivo synthesis of l‐[13C6]kynurenine and [13C6]kynurenate from l‐[13C6]tryptophan was studied in the CSF of macaques infected with poliovirus, as a model of inflammatory neurologic disease. The effects of 6‐chlorotryptophan and 4‐chloro‐3‐hydroxyanthranilate on the synthesis of kynurenate were different. 6‐Chlorotryptophan attenuated formation of l‐[13C6]kynurenine and [13C6]kynurenate and was converted to 4‐chlorokynurenine and 7‐chlorokynurenate. It may be an effective prodrug for the delivery of 7‐chlorokynurenate, which is a potent antagonist of NMDA receptors. In contrast, 4‐chloro‐3‐hydroxyanthranilate did not reduce accumulation of l‐[13C6]kynurenine and [13C6]kynurenate. 6‐Chlorotryptophan and 4‐chloro‐3‐hydroxyanthranilate are useful tools to manipulate concentrations of quinolinate and kynurenate in the animal models of neurologic disease to evaluate physiological roles of these neuroactive metabolites.