Synthesis and release of neurotoxic kynurenine metabolites by human monocyte-derived macrophages

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Abstract

We studied the regulation of the kynurenine pathway of tryptophan metabolism in human monocyte-derived macrophages (MDM) with the aim of evaluating macrophage involvement in inflammatory neurological disorders.

Cultured MDM metabolized tryptophan and released kynurenine metabolites, including the excitotoxin quinolinic acid (QUIN). Lipopolysaccharides (LPS) or the pro-inflammatory cytokines INFγ and TNFα increased, while IL 4 or IL 10 inhibited the rate of tryptophan metabolism and the release of QUIN.

The incubation media of INFγ-exposed MDM caused neuronal death in primary cultures of mixed cortical cells. Glutamate receptor antagonists or poly(ADP-ribose) polymerase inhibitors significantly reduced this death, thus suggesting new possibilities for the treatment of neuronal damage in neuroinflammatory disorders.

Introduction

It has been repeatedly shown that several metabolites formed along the “kynurenine pathway” of tryptophan metabolism may affect neuronal function and survival. These include 3-hydroxy-kynurenine (3OH-KYN), a neurotoxin able to induce oxidative stress and apoptotic neuronal death Okuda et al., 1998, Moroni, 1999, and quinolinic acid (QUIN), a potent NMDA receptor agonist and excitotoxic agent Perkins and Stone, 1982, Schwarcz et al., 1983.

Most of the enzymes of the “kynurenine pathway” are present in macrophages (Heyes et al., 1992b) where they may be induced by bacterial lipopolysaccharides (LPS) or by pro-inflammatory cytokines such as interferon γ (INFγ) and tumor necrosis factor α (TNFα) Saito et al., 1992, Alberati-Giani et al., 1996b, Pemberton et al., 1997, Chiarugi et al., 2000b. The biological meaning of this induction remains to be clarified. It has been proposed, however, that an increased rate of tryptophan catabolism may lead to local depletion of this amino acid, an event associated with a reduced replication rate of intracellular pathogens such as Toxoplasma or Chlamydia Pfefferkorn, 1984, Carlin et al., 1989. It has also been proposed that an increased rate of tryptophan metabolism in human monocyte-derived macrophages (MDM) may inhibit the proliferation of neighboring cells (including T lymphocytes) and that this immuno-suppressive mechanism is particularly important in explaining the tolerance of the mother towards the fetus Munn et al., 1998, Mellor and Munn, 1999.

In the central nervous system (CNS), however, this immunoregulatory mechanism may be dangerous because a significant increase in the rate of tryptophan metabolism leads to local accumulation of neurotoxic tryptophan metabolites. This accumulation has been found in AIDS-related dementia complex Heyes et al., 1991, Saito et al., 1992, multiple sclerosis Panitch, 1992, Chiarugi et al., 2000a, measles or malaria encephalitis Andersson et al., 1993, Sanni et al., 1998, global or focal brain ischemia Saito et al., 1993, Cozzi et al., 1999 and brain or spinal cord trauma (Blight et al., 1995).

In order to gain a better understanding of the regulation of the kynurenine pathway, we exposed human MDM to pro- and anti-inflammatory cytokines. We measured the activity of kynurenine pathway enzymes in activated cultures as well as the concentration of tryptophan and its metabolites in the incubation media. We also exposed primary cultures of mixed cortical cells to MDM-conditioned media in order to evaluate the neurotoxic potential of macrophage-derived compounds. Our results showed that, in cultured MDM, the activities of the kynurenine pathway enzymes are finely regulated by pro- and anti-inflammatory cytokines. They also showed that cultured neurons exposed to MDM-conditioned media undergo excitotoxic cell death.

Section snippets

Chemicals

Human recombinant INFγ, TNFα, interleukin (IL) 4 and IL10 were from PeproTech EC (London, UK). [713C]-QUIN was provided by Dr. J.F. Reinhard Jr. (Research Triangle Park, NC, USA). Purified kynurenine monooxygenase (KMO) and mouse monoclonal antibodies directed to KMO (clone Fpk7c1) (Uemura and Hirai, 1998) were a generous gift of Dr. T. Uemura (Setagaya-ku, Tokyo). Tryptophan, kynurenine (KYN), 3OH-KYN, anthranilic acid (ANA), QUIN and LPS (Escherichia coli) were from Sigma (St. Louis, MO,

Tryptophan metabolism in MDM: effects of LPS and cytokines

Cultured MDM metabolized tryptophan and released KYN, ANA and QUIN. LPS (1 μg/ml), INFγ (100 U/ml) and TNFα (10 ng/ml) significantly increased the rate of this metabolism and the release of KYN, ANA and QUIN. Surprisingly, however, neither LPS nor the cytokines increased the release of 3OH-KYN, a toxin able to cause neuronal apoptosis (see Fig. 1).

In separate experiments, we tested the effects of the inhibitory cytokines IL-4 or IL-10 (both at 10 ng/ml) on KYN and QUIN output. Table 1 shows

Discussion

Our experiments show that activated human MDM in culture rapidly metabolize tryptophan and locally release a number of kynurenine metabolites, including the excitotoxin QUIN. LPS and pro-inflammatory cytokines, such as TNFα or INFγ, significantly increase, while other cytokines such as IL 4 or IL 10, inhibit the rate of tryptophan metabolism and the release of kynurenine metabolites. The experiments also show that MDM-conditioned media, applied to primary cultures of mixed cortical cells, cause

Acknowledgements

This work was supported by grants from the Istituto Superiore di Sanità (Multiple Sclerosis research project), the University of Florence, MURST and CNR. We would like to thank Dr. J.F. Reinhard Jr. for providing [713C] quinolinic acid and Dr. T. Uemura for the kind gift of purified kynurenine monooxygenase and anti-kynurenine monooxygenase antibodies.

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