kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection: kynurenine...

UNCORRECTED PROOF

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1±13 1

Journal of Neurochemistry, 2001, 78, 1±13

Kynurenine pathway metabolism in human astrocytes: a paradox

for neuronal protection

Gilles J. Guillemin,* Stephen J. Kerr,* George A. Smythe,³ Danielle G. Smith,* Vimal Kapoor,§Patricia J. Armati,¶ Juliana Croitoru** and Bruce J. Brew*,²

*Centre for Immunology and ²Departments of Neurology and HIV Medicine, St. Vincent's Hospital, Sydney, Australia

³The Ray Williams Biomedical Mass Spectrometry Facility and §School of Physiology and Pharmacology, Faculty of Medicine,

University of New South Wales, Sydney, Australia

School of Biological Sciences, University of Sydney, Australia

**Commissariat a l'Energie Atomique, Department of Neurovirology, Fontenay-aux-Roses, France

Abstract

There is good evidence that the kynurenine pathway (KP) and

one of its products, quinolinic acid (QUIN), play a role in the

pathogenesis of neurological diseases, in particular AIDS

dementia complex. Although QUIN has been shown to be

produced in neurotoxic concentrations by macrophages and

microglia, the role of astrocytes in QUIN production is

controversial. Using cytokine-stimulated culture of human

astrocytes, we assayed key enzymes and products of the KP.

We found that human astrocytes lack kynurenine hydroxylase

so that large amounts of kynurenine and the QUIN antagonist

kynurenic acid were produced. In contrast, the amounts of

QUIN that were synthesized were subsequently completely

degraded. We then showed that kynurenine in concentrations

comparable with those produced by astrocytes led to

signi®cant production of QUIN by macrophages. These results

suggest that astrocytes alone are neuroprotective by mini-

mizing QUIN production and maximizing synthesis of kynure-

nic acid. However, it is likely that, in the presence of

macrophages and/or microglia, astrocytes become indirectly

neurotoxic by the production of large concentrations of

kynurenine that can be secondarily metabolized by neighbour-

ing or in®ltrating monocytic cells to form the neurotoxin QUIN.

Keywords: human astrocyte, kynurenine pathway, kynure-

nine, quinolinic acid, neurotoxicity.

J. Neurochem. (2001) 78, 1±13.

There is good evidence that the kynurenine pathway (KP) is

involved in the neurocytotoxicity associated with AIDS

dementia complex and probably other in¯ammatory brain

diseases (Heyes 1996). The KP is a major route of

l-tryptophan catabolism, resulting in the production of

nicotinamide adenine dinucleotide and other neuroactive

intermediates (Fig. 1) (Bender and McCreanor 1982; Stone

1993; Botting 1995; Curzon 1996; Moroni 1999). These

include kynurenine (KYN) (Lapin et al. 1982), kynurenic

acid (KYNA), 3-hydroxykynurenine (3-HK) (Eastman 1989;

Moroni 1999), picolinic acid (PIC) (Melillo et al. 1996) and

quinolinic acid (QUIN) (Lapin et al. 1982; Schwarcz et al.

1983; Stone 1993). Of the metabolites, the NMDA receptor

agonist and neurotoxin QUIN is the most important.

Moreover, the KP may play a role in certain physiological

functions (Stone 1993; Curzon 1996) such as behaviour,

sleep, thermoregulation and pregnancy (Munn et al. 1998).

The cellular localization of the KP has been shown to be

primarily in macrophages and microglial cells (Heyes et al.

1996; Espey et al. 1997) and possibly in astrocytes (Moffett

et al. 1993; Saito et al. 1993b; Speciale and Schwarcz 1993;

Received April 19, 2001; revised manuscript received May 18, 2001;

accepted May 27, 2001.

Address correspondence and reprint requests to Gilles Guillemin,

Centre for Immunology, St. Vincent's Hospital, Darlinghurst, 2010

Sydney, NSW, Australia. E-mail: g.guillemin@c®.UNSW.edu.au

Abbreviations used: 3-HAA, 3-hydroxyanthranilic acid; 3-HAO,

3-hydroxyanthranilate dioxygenase; 3-HK, 3-hydroxykynurenine;

GC-MS, gas chromatography±mass spectrometry; GFAP, glial ®brillary

acid protein; GM-CSF, granulocyte2macrophage colony-stimulating

factor; IDO, indoleamine 2,3-dioxygenase; IFN, interferon; IL, inter-

leukin; KAT, kynurenine amino transferase; KP, kynurenine pathway;

KYN, kynurenine; KYNA, kynurenic acid; KYNase, kynureninase;

KYN-OHase, kynurenine hydroxylase; MdM, blood monocyte derived

macrophages; PBS, phosphate-buffered saline; PIC, picolinic acid;

QPRTase, quinolinate phosphoribosyltransferase; QUIN, quinolinic

acid; TNF, tumour necrosis factor.

JNC498 JNC jn6489 CD 12/7/1 12:38 ALDEN

UNCORRECTED PROOF

Heyes et al. 1996, 1997a, b; Espey et al. 1997). In support of

the latter, increased indoleamine 2,3-dioxygenase (IDO)

activity has been found in astrocytes (Moffett et al. 1993;

Speciale and Schwarcz 1993; Heyes et al. 1997a, b), but it is

not clear whether the other enzymes in the KP are present

and functional in man (Schwarcz et al. 1983; Okuno et al.

1987; KoÈhler et al. 1988b; Du et al. 1992; Heyes et al. 1992,

1997; Heyes 1993; Saito et al. 1993a, b; Stone 1993;

Guidetti et al. 1997; Sanni et al. 1998; Tamburin et al.

1999).

To de®ne the KP in human astrocytes, we used human

fetal astrocyte cultures stimulated with some of the

cytokines involved in brain in¯ammation, especially those

associated with AIDS dementia complex, namely, inter-

leukin (IL)-1b, IL-6, granulocyte2macrophage colony

stimulating factor (GM-CSF), interferon (IFN)-g and

tumour necrosis factor (TNF)-a. Although IFN-g and

TNF-a are known to induce the ®rst enzyme of the KP

(IDO) (Heyes et al. 1997a; Pemberton et al. 1997), their role

in stimulating the other KP enzymes is unde®ned. We

therefore designed primer sets for seven of the major KP

enzymes, namely, IDO (EC 1.13.11.17), kynurenine-

pyruvate aminotransferase (KAT-I; EC 2.6.1.64), kynure-

nine-2-oxoglutarate aminotransferase (KAT-II; EC 2.6.1.7),

kynurenine hydroxylase (KYN-OHase; EC 1.14.13.9),

kynureninase (KYNase; EC 3.7.1.3), 3-hydroxyanthranilate

dioxygenase (3-HAO; EC 1.13.11.6) and quinolinate

phosphoribosyltransferase (QPRTase; EC 2.4.2.19). Expres-

sion of these enzymes was then assessed by RT-PCR. In

addition, we measured the concentration of important KP

intermediates KYN and KYNA and end-products QUIN and

picolinic acid.

Materials and methods

Reagents and chemicals

All cell culture media and additives were from Life Technologies

(Gaithersburg, MA, USA) unless otherwise stated. TNF-a, IL-1b,

IL-6, IFN-g and mouse monoclonal antibody anti-MAP2 came

from Boehringer Mannheim (Mannheim, Germany). GM-CSF was

kindly provided by Schering-Plough (Sydney, NSW, Australia).

3-HAA, KYN, PIC, QUIN, polyclonal antibody antigalactocerebro-

side protein and DAPI were obtained from Sigma Chemical Co.

(Sydney, NSW, Australia). Anti-mouse IgG1 antibodies coupled to

magnetic microbeads were obtained from Dynal (Carlton, VIC,

Australia). Mouse monoclonal antibody anti-glial ®brillary acid

protein (GFAP) clone GA-5 was obtained from Novacostra

(Newcastle, UK). Mouse monoclonal antibody anti-CD68 clone

KiM1P was gratefully provided by Dr Parwaresch and was used at

a concentration of 10 mg/mL. Mouse monoclonal antibody anti-

CD14 clone TUK-4 and mAb anti-factor VIII clone F8/86 were

from Dako (Carpintera, CA, USA). Secondary goat anti-mouse

IgG1 and anti-rabbit ¯uorescein isothiocyanate or Texas Red-

conjugated antibodies were purchased from Southern Biotech-

nology Associates (Birmingham, AL, USA). All commercial

Fig. 1 Simpli®ed kynurenine pathway in

astrocytes.

Q1

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2 G. J. Guillemin et al.

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1±13

UNCORRECTED PROOF

antibodies were used at the concentrations recommended by the

manufacturer.

Cell cultures

Human fetal brains were obtained from 14218-week-old aborted

fetuses collected after therapeutic termination following informed

consent. Astrocytes were prepared using a protocol adapted from

previously described methods (Guillemin et al. 1997; Kerr et al.

1997). Brie¯y, cerebral portions were washed thoroughly with

phosphate-buffered saline (PBS), forced through a 100-mm nylon

mesh with the plunger of a plastic syringe. The suspension was

centrifuged at 500 g for 5 min and the cell pellet resuspended in

RPMI-1640 medium containing 10% heat-inactivated fetal calf

serum, 2 mm glutamine, 200 IU/mL penicillin G, 200 mg/mL

streptomycin sulfate and 0.5% glucose, then plated onto 75 cm2

culture ¯asks (Corning, NY, USA). Cultures were kept at 378C at

5% CO2 in a humidi®ed atmosphere. After 3 days, medium was

changed at 5 and 10 days. The cells became con¯uent by 10±12

days. Microglia were removed from the cultures by gently shaking

¯asks for 2 h at 220 r.p.m. at room temperature (228C) and the

medium changed, thereby removing ¯oating cells. A second

puri®cation step was performed to ensure that highly pure astrocyte

cultures were obtained by using immunomagnetic depletion of

monocyte-lineage cells using the monoclonal antibodies anti-CD14

and anti-CD68 coupled to magnetic microbeads. The remaining

cells were rinsed twice with PBS and cultured as above in non-

coated ¯asks with the culture medium and maintained for up to 6

weeks. The medium was changed twice a week. Blood monocyte-

derived macrophages (MdM) were obtained from human volunteers

(Centre for Immunology, Sydney, Australia) using a classic

adherence method (Kerr et al. 1997).

Immunocytochemistry

The method for brain cell characterization has been described

previously (Guillemin 1997). The following three controls were

performed for each labelling experiment: (i) isotypic antibody

controls, (ii) incubation with only the secondary labelled anti-

bodies, and (iii) estimation of auto¯uorescence of unlabelled cells.

Astrocyte culture treatments

Based on standard curve optimizations (data not shown), 1 mm

QUIN or 100 mm of 3-HAA was added to 4-week-old pure

astroglial cultures in the presence or absence of 100 IU/mL IL-1b,

IL-6, TNF-a, GM-CSF or IFN-g. RNA and culture supernatants

were collected after 6, 24, 48 and 72 h. Each experiment was

performed in triplicate using cultures derived from three different

fetal brains.

Macrophage culture treatments

Either 12.5 or 50 mm of KYN was added to 10-day-old MdM

cultures stimulated or not with 100 IU/mL IFN-g. MdM culture

stimulated with 100 IU/mL IFN-g was used as positive controls for

IDO activation and QUIN production. Culture medium AIM-Vw

(serum-free medium) was used for these experiments. Supernatants

were collected after 24 and 48 h incubation, and RNA after 48 h.

Experiments were performed in triplicate on cultures derived from

three different fetal brains.

Mass spectrometry

Culture supernatants were assayed for QUIN as described

previously (Kerr et al. 1997). QUIN levels were calculated using

the formula [Total nm of QUIN detected ± nm of QUIN present in

the 10% fetal calf serum culture medium] or the levels of QUIN

effectively catabolized after 48 h. The levels were calculated by the

following formula: [Total nm of QUIN detected ± 1000 nm of

QUIN added in the medium ± nm present in the 10% fetal calf

serum culture medium]. Following the same derivatization

methodology, samples were prepared for PIC analysis using

d4-picolinic acid as an internal standard. QUIN and PIC samples

were analysed by GC-MS with the spectrometer operating in

electron capture negative ionization mode. Selected ions (m/z 273

for PIC and m/z 277 for d4-PIC) were then monitored (Smythe,

Kerr, Guillemin and Brew, manuscript submitted). Effective

PIC de novo synthesis was calculated using the formula [PIC in

sample ± PIC in culture medium]. All results are expressed as the

mean ^SEM.

HPLC

Kynurenine was measured by reverse-phase HPLC with UV

detection at 365 nm, using an ammonium acetate buffer (0.1 m,

pH 4.65) containing 0.02% acetonitrile as the organic modi®er. The

limit of detection was estimated to be 0.6 mm of KYN [Total nm of

KYN detected ± nm of KYN present in the 10% fetal calf serum

culture medium]. Kynurenic acid was measured by reverse-phase

HPLC with ¯uorescence detection after post column derivatization

with zinc acetate as described previously (Kapoor et al. 1994). The

limit of detection was estimated to be 10 fm of KYNA. Amounts of

KYNA were calculated with the formula [Total nm of KYNA

detected ± nm of KYNA present in the 10% fetal calf serum culture

medium]. HPLC with electrochemical detection was used for the

quantitation of 3-hydroxykynurenine (3-HK) essentially according

to the method of Heyes et al. (1998). The limit of detection was

estimated at 0.1 nm of 3-HK. All results are expressed as the

mean ^SEM.

RT-PCR detection of mRNA expression of KP enzymes

cDNAs were synthesized for 1 h at 458C from 1 mg of RNA

extracted using Trizolw reagent (Life Technologies, Mulgrave,

Australia), in a 100-mL ®nal volume of 10 mm Tris2HCl (pH 8.3),

25 mm KCl, 0.6 mm MgCl2, 1 mm dNTP, 5 U/mL of Avian

Myeloblastosis virus reverse transcriptase (Promega, Madison, WI,

USA), 0.01 mg/mL of oligo(dT)15 (Boehringer), 2 U/mL of RNasin

(Promega). PCR was performed on 1 mg of cDNA in a 50-mL

reaction mixture containing 10 mm Tris2HCl pH 8.3, 50 mm KCl,

1.5 mm MgCl2, 10 mm of dNTPs, 0.25 mm of speci®c primers

(Geneworks, Sydney, Australia), and 1.25 U/mL Taq DNA

polymerase (Boehringer). Individual sets of primers were used to

identify mRNA transcripts for human IDO, KAT-I, KAT-II,

KYNase, KYN-OHase, 3-HAO and QPRTase, and spanned at

least one intron to distinguish ampli®cation of cDNA from genomic

DNA. Primers were designed using the application Primers! for the

Mac v1.0a (q1996, Apple Pi, Ashland, MA, USA). Amplify 1.2

(William Engels, Genetics Department, University of Wisconsin,

Madison, WI, USA) was used to estimate the number and size of

PCR products. KP enzyme gene sequences were obtained from

GenBank, see Table 1. Reporter gene GAPDH primers have been

described previously (Villinger et al. 1993). Previously published

primers for IDO (Koide and Yoshida 1994) were used to con®rm

our IDO results (data not shown), and sequences of PCR products

were veri®ed for all primer sets. PCR conditions and cycling

Q3

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Kynurenine pathway in astrocytes 3


UNCORRECTED PROOF

parameters were optimized using primary human fetal astrocytes

stimulated with 100 IU/mL IFN-g (data not shown). Negative

controls were: (i) omission of a target template, (ii) omission of

reverse transcriptase, and (iii) genomic DNA. PCR was performed

in an eppendorf gradient cycler (Hamburg, Germany) according to

the following protocol: 35 cycles at 928C for 1 min, 608C for

1 min, and 728C for 90 s. PCR products were loaded in 2%

agarose/1 mg/mL ethidium bromide gel. Ampli®ed products were

visualized under UV light, and quanti®ed after scanning using nih

image 1.61 (NIH, Bethesda, MA, USA). Experiments were

performed in duplicate on cultures derived from three different

fetal brains. Based on image analysis intensity ratios of KP enzyme

mRNA expressed relative to GAPDH mRNA, the standard error

was between 4 and 5%.

Results

Purity of primary cultures of human fetal astrocytes

Cultures of fetal brain cells immunostained with the

astrocytic marker GFAP were 90±95% astrocytic after 7

days in culture and . 99% pure after 1 month of culture

(Fig. 2a). Polyclonal or monoclonal antibodies tested

against galactocerebroside protein (oligodendrocytes),

MAP2 (neurons), factor VIII (endothelial cells) did not

stain any of the cells at 15 or 30 days after seeding.

Monoclonal antibodies directed against CD14 and CD68

proteins showed that , 5% of cells were microglia (Fig. 2b),

Table 1 Sequences of primer sets used to detect KP enzyme expression

Genes Primers Sequences

hu-IDO Forward 5 0-ACCACAAGTCACAGCGCC-3 0

Reverse 5 0-CCCAGCAGGACGTCAAAG-3 0

hu-KAT-1 Forward 5 0-TCGAACCCTTTTTTGACTGC-3 0

Reverse 5 0-CTGCTCCCGTTCAAAGCTCT-3 0

hu-KAT-2 Forward 5 0-AGCTGGCATGTTTCTATGGA-3 0

Reverse 5 0-AAGTGGTTCAGGGAAATTGC-3 0

hu-KYN-OHase Forward 5 0-GATGAGGAAGATAAGCTGAGGC-3 0

Reverse 5 0-CTTAAGGTTTCTTCCCCCTCTC-3 0

hu-KYNase Forward 5 0-GACTATTCCACCTAAGAACGGAGA-3 0

Reverse 5 0-ACAGGAAGACACAAACTAAGGTCG- 3 0

hu-3HAO Forward 5 0-GAGGGCCTTCTTGGTGTG-3 0

Reverse 5 0-CATTGATTGGTTGTGAGTGG-3 0

hu-QPRTase Forward 5 0-GGTCACAGGAGCAGCAGG-3 0

Reverse 5 0-AAGCCAGAGGAGCTGCAC-3 0

Gene sequences have been obtained from GenBank (Internet) and primers have been calculated using Primers for the Mac v1.0a (q1996, Apple

Pi, Ashland, USA).

Fig. 2 Purity of primary cultures of

human fetal astrocytes. (a) GFAP immu-

nocytochemical staining of puri®ed human

primary astrocyte cultures (�400). (b) CD68

immunocytochemical staining of isolated

human primary microglial cells removed

from astrocyte cultures (�400).




UNCORRECTED PROOF

however, shaking, immunodepletion and successive trypsi-

nizations led to their total disappearance so that after 3±5

weeks of culture, only astrocytes were present in the

cultures.

Kynurenine and kynurenic acid, but not

3-hydroxykynurenine, are synthesized by puri®ed

cultures of human fetal astrocytes

Kynurenine was not detectable in the culture medium

(containing 10% fetal calf serum). The concentration of

KYNA in the medium was 85.5 ^ 5.1 nm. Unstimulated

astrocytes did not produce detectable amounts of KYN and

only minor amounts of KYNA (Fig. 3a and b). IFN-g

(100 IU/mL) and IFN-g plus TNF-a (100 IU/mL) induced

the production of 11.7 ^ 1.5 and 10.3 ^ 0.9 mm KYN,

respectively (Fig. 3a). Other cytokines such as TNF-a,

IL-1b, IL-6, GM-CSF (100 IU/mL) and QUIN (350 nm)

were not able to induce any detectable KYN production

(limit of detection 0.6 mm). Only IFN-g induced a

signi®cant increase in KYNA synthesis with KYNA levels

of 60.5 ^ 14.7 nm for IFN-g (100 IU/mL) and 25 ^ 8.1 nm

for IFN-g plus TNF-a (100 IU/mL) (Fig. 3b). However,

TNF-a strongly decreased the stimulatory effect of IFN-g

on KYNA synthesis. Control cultures minus cytokine

treatment synthesized only 13.2 ^ 5.3 nm KYNA. Final

KYNA concentrations were calculated after subtraction of

the amount of KYNA detected in the culture medium. 3-HK

synthesis was not detected in any of the astrocyte cultures

whatever the treatment with cytokines.

Human fetal astrocytes produced low levels of QUIN,

which were rapidly degraded

In the ®rst experiment, no 3-HAA or QUIN was added to the

culture (Fig. 4a; control without 3-HAA or QUIN). QUIN

concentration in the culture medium (with 10% fetal calf

serum) at the beginning of the experiment was

354.7 ^ 5.1 nm. After 48 h, the non-stimulated astrocytes

(control) degraded the low levels of QUIN with

Fig. 3 Quanti®cation of the in vitro de

novo synthesis of KYN in mM (a) and

KYNA in nM (b) by primary human fetal

astrocytes after 48 h, following various

cytokine treatments (as described in

Materials and methods). *p , 0.002 and

**p , 0.01 compared with controls (Mann2

Whitney test).

Fig. 4 In vitro de novo synthesis or

catabolism of QUIN by primary human

fetal astrocytes. Quanti®cation of QUIN

production or degradation compared with

culture medium with fetal calf serum after

48 h (as described in Materials and meth-

ods); without addition of 3-HAA or QUIN

(a), or following addition of 100 mM 3-HAA

(b), or 1 mM QUIN itself (c) to the culture

medium and various cytokine treatments.

*Signi®cant compared with respective `Con-

trol' (no cytokine; p , 0.02 (Mann2Whitney

test).




UNCORRECTED PROOF

150.24 ^ 7.4 nm less QUIN compared with levels detected

at the beginning of the experiment. The addition of 100 IU/

mL of IL-1b, IL-6 or GM-CSF increased QUIN catabolism

by astrocytes signi®cantly (Fig. 4a). The levels of QUIN

were, respectively, 191.94 ^ 4.1 (with IL-1b), 175.9 ^ 9.9

(with GM-CSF) and 202.63 ^ 15.7 nm (with IL-6).

When 100 mm of 3-HAA was added to the culture

medium with or without 100 IU/mL of IL-1b, IL-6, TNF-a

or GM-CSF (Fig. 4b), only low levels of QUIN production

were observed, indicating that under these conditions

astrocytic QUIN production can exceed catabolism. After

48 h, the mean concentrations of QUIN detected in the

culture supernatants were between 3.21 ^ 29.0 and

31.01 ^ 2.14 nm (average 14.27 nm). No signi®cant varia-

tion was observed following cytokine treatments. When

cultured astrocytes were exposed to IFN-g QUIN catabolism

was higher than QUIN synthesis (39.03 ^ 6.9 nm).

When 1 mm of QUIN was added to the cultures in the

presence or absence of 100 IU/mL of TNF-a, IL-1b, IL-6,

GM-CSF or IFN-g (Fig. 4c). This signi®cant increase in

QUIN catabolism was induced in comparison with the

control cultures (296.7 ^ 9.3 nm). QUIN catabolism was

between 296.7 ^ 9.3 (control), 580.9 ^ 56.1 (IL-6) and

560.1 ^ 42.2 nm (IL-1b) under these conditions.

Fetal astrocytes can produce picolinic acid

A new and speci®c GC-MS method was developed and

optimized to quantify PIC (Smythe et al., manuscript

submitted). After 48 h, non-stimulated astrocytes produced

PIC (13.4 ^ 8 nm) (Fig. 5). Astrocytes exposed to IL-6

produced only minor amounts (24.3 ^ 8 nm) of PIC. In

contrast, IFN-g strongly inhibited PIC synthesis to a level of

only 1.6 ^ 0.7 nm.

In the presence of 100 mm of 3-HAA, no signi®cant

variation of PIC concentrations was observed. IL-6-treated

cultures with 3-HAA produced the same amount of PIC of

24.3 ^ 8.3 nm as those without 3-HAA. Moreover, 3-HAA

fully reverted the inhibitory effect of IFN-g on PIC synthesis

(19.8 ^ 1.3 nm).

Expression of KP enzymes in human fetal astrocytes

Initially, original primer pairs were developed to identify

mRNA transcripts for human IDO, KAT-I, KAT-II, KYN-

OHase, KYNase, 3-HAO and QPRTase (see Materials and

methods). Based on image analysis intensity ratios of KP

enzyme mRNA expressed relative to GAPDH mRNA, the

standard error was between 4 and 5% (data not shown).

IFN-g was the only cytokine able to induce IDO mRNA

expression in primary human astrocyte cultures, whereas

Fig. 5 In vitro de novo synthesis of PIC

by primary fetal astrocytes. Quanti®cation

of PIC synthesis after 48 h (as described in

Materials and methods) following addition of

100 mM of 3-HAA to the culture medium

and various cytokine treatments. *p , 0.02

compared with control (Mann2Whitney

test).

Fig. 6 Expression of KP enzymes.

Photograph of ethidium bromide-stained gel

showing RT-PCR for KP enzyme expres-

sion, 24 h after cytokine treatments in the

presence and absence of 3-HAA or QUIN.

Human primary macrophages (Mù) were

used as a positive control for enzyme

expression.

Q5




UNCORRECTED PROOF

IL-1b, IL-6, TNF-a and GM-CSF had no effect on IDO

induction (Fig. 6). Interestingly, IDO mRNA expression in

response to IFN-g could be delayed temporarily by the

addition of 100 mm 3-HAA or 1 mm QUIN to the culture

medium (data not shown).

Unstimulated astrocytes expressed both KAT-I and

KAT-II. However, expression of KAT-I was signi®cantly

higher than KAT-II (30�). KAT subtype mRNA expression

was not modi®ed by treatment with 100 IU/mL of IL-1b,

IL-6, TNF-a or GM-CSF. Interestingly, IFN-g was able to

potentiate mRNA expression of both KAT subtypes. 3-HAA

or QUIN increased KAT-I mRNA expression signi®cantly

expression of KAT-II mRNA to a lesser extent.

KYNase mRNA expression was strongly potentiated by

the presence of 3-HAA or QUIN. No signi®cant variations

were observed following cytokine treatment.

KYN-OHase mRNA expression was not detected in

primary astrocyte cultures regardless of cytokine treatment.

Primary astrocytes were found to constitutively express

3-HAO. This expression was not altered by any cytokine

treatment, and only weakly increased by the presence of

3-HAA or QUIN in the culture medium.

As for KYNase, QPRTase mRNA expression was highly

potentiated by the presence of 3-HAA or QUIN and no

signi®cant variations were observed following treatment

with 100 IU/mL of IL-1b, IL-6, TNF-a or GM-CSF.

However, QPRTase mRNA expression was signi®cantly

increased by IFN-g.

IFN-g-stimulated macrophages were used as a positive

control. For all the enzymes, mRNA expression was

analysed at 6, 24, 48, 72 h (only 24 h data are presented).

A maximal mRNA expression rate was obtained at 24 h. All

experiments were carried out in triplicate as above.

Kynurenine increases QUIN production and KP enzyme

expression by human macrophages

The presence of KYN 12.5 mm and 50 mm increased both

KYNase, KYN-OHase expression (Fig. 7a) and QUIN

production by MdM (Fig. 7b). KYN combined with IFN-g

(100 IU/mL) induced IDO, KYNase and KYN-OHase

expression (Fig. 7a) leading to a higher and signi®cant

QUIN synthesis than KYN 12.5 mm and 50 mm alone

(Fig. 7b). AIM-Vw medium and IFN-g-stimulated macro-

phages were used, respectively, as negative and positive

controls. Each experiment was carried out in triplicate using

macrophages from three different blood donors.

Discussion

This study provides the ®rst comprehensive analysis of the

KP in human astrocytes with implications for the patho-

genesis of in¯ammatory neurological diseases. We have

demonstrated for the ®rst time that KYN-OHase, a critical

enzyme in the KP, is absent from human astrocytes in vitro,

thereby explaining our ®nding that astrocytes cannot

produce 3-HK but are able to produce large amounts of

the early KP metabolites, such as KYN and KYNA, and

only minute amounts of the late metabolites QUIN (only

when 3-HAA is added) and PIC. Essentially, the KP is

`split' in half in astrocytes (Fig. 1) compared with macro-

phages, which have the full complement of KP metabolites.

Moreover, we have shown that the ®rst enzyme in the KP,

IDO, can be induced by IFN-g but not by the other cytokines

and leads to further excess production of these early

Fig. 7 Presence of kynurenine increased QUIN production by

macrophages. (a) Agarose gel showing RT-PCR for KP enzyme

expression 48 h after treatment by kynurenine (12.5 and 50 mM) with

or without IFN-g (100 U/ml). (b) QUIN production measured by

GC-MS. *p , 0.01 compared with control AIM-Vw; ³p , 0.02 com-

pared with control KYN 12.5 mM; 1p , 0.05 compared with control

KYN 50 mM (Mann2Whitney test).




UNCORRECTED PROOF

metabolites. Although there is overproduction of early

metabolites, the amounts of KYNA, an antagonist of

QUIN, are 200-fold less than those of KYN, probably

secondary to a 30-fold lower expression of KAT-II

compared with KAT-I. Our results also demonstrate that

the production of the late metabolites was more complex.

Astrocytes express an ef®cient IFN-g-inducible QPRTase,

which explains our ®nding that astrocytes degrade QUIN.

However, we also showed that the excess of KYN,

synthesized by the cytokine-stimulated astrocytes could be

metabolized by macrophages to produce signi®cant con-

centrations of QUIN. These ®ndings have implications not

only for AIDS dementia complex, but also for other

in¯ammatory conditions of the human CNS.

Until now, the capacity of astrocytes to produce QUIN

and other KP metabolites has been controversial and the KP

enzymes within astrocytes have not been delineated in full.

Of particular interest under our culture conditions with

puri®ed cells, human fetal astrocytes were primarily

associated with QUIN catabolism rather than synthesis. In

contrast, in rat brain, several biochemical (Schwarcz et al.

1988a) and immunocytochemical (KoÈhler et al. 1988a, b)

studies have shown that QUIN production from 3-HAA can

take place within astrocytes (Speciale and Schwarcz 1993)

and injection of l-KYN (Guidetti et al. 1995) or 3-HAA

(Heyes and Markey 1988; Speciale and Schwarcz 1993)

induces an increase in QUIN production. The controversial

issue regarding astrocytic participation in the in¯ammatory/

neurotoxic process arises from con¯icting results from other

studies of rat and human brain cell cultures and gerbil

astrocytes. For example, either human fetal brain cell

cultures enriched in astrocytes (Heyes et al. 1997a) or

activated gerbil astrocytes (Heyes et al. 1997b) have the

capacity to produce l-KYN and QUIN but the levels

produced are very low compared with microglia or

macrophages (Heyes et al. 1996; Espey et al. 1997).

Conversely, other studies in rat brain have shown that

QUIN immunoreactivity is only detectable in activated

macrophages/microglia but not in reactive astrocytes

(Moffett et al. 1997). Also, in rat primary mixed brain cell

cultures composed of 60% astrocytes, no QUIN production

was detected after 3-HAA was added (Heyes et al. 1997a).

Thus, our ®nding that puri®ed primary cultures of human

fetal astrocytes can produce small amounts of QUIN is in

agreement with other reports (KoÈhler et al. 1988b, c;

Schwarcz et al. 1988a; Speciale and Schwarcz 1993; Heyes

et al. 1996, 1997a). The important difference in our system

is that this production occurred only after 3-HAA was added

to the culture medium, a precursor not normally available to

brain tissue (Saito et al. 1993a). These con¯icting results

may well re¯ect species differences but could also relate to

both the relative insensitivity of immunocytochemical

methods and the use of mixed brain cell cultures rather

than puri®ed primary cultured human astrocytes.

Our results for the expression of KP enzymes in human

astrocytes con®rm and extend previous studies (Okuno et al.

1987; KoÈhler et al. 1988a, b; Schwarcz et al. 1988a; Du et al.

1991; Roberts et al. 1992) in that IDO is present in these

astrocytes and is inducible by IFN-g (Werner-Felmayer et al.

1989; Saito et al. 1993b; Alberati-Giani et al. 1996; Heyes

et al. 1996, 1997a). However, in contrast with studies

employing macrophages (Heyes et al. 1997a; Pemberton

et al. 1997), TNF-a did not activate IDO or induce QUIN

production.

Our results concerning KYNase and KYN-OHase are

only partly in accord with published data. We have shown

that KYNase is expressed by isolated human astrocytes and

3-HAA or QUIN potentiate this expression. Biochemical

studies by others, however, have shown that KYNase

activity is very low in astrocytoma cell lines and not

detectable in human fetal brain cell cultures (Heyes et al.

1996, 1997a). KYN-OHase has recently been detected in

adult rat neurons and astrocytes using immunohistochem-

istry (Chiarugi et al. 2001) but KYN-OHase activity is

undetectable in either human astrocytoma or human fetal

brain cell cultures (Heyes et al. 1996, 1997a). However, we

demonstrated that KYN-OHase expression is absent in

human fetal astrocytes in accord with existing literature.

Moreover, we did not detect any expression of KYN-OHase

mRNA in puri®ed and concentrated mitochondrial fraction

from human fetal astrocytes (Almeida and Medina 1998)

stimulated or not with IFN-g (data not shown). These results

were con®rmed by the lack of production of 3-HK, a KP

metabolite able to potentiate QUIN neurotoxicity (Eastman

1989). Thus, we propose that 3-HAA is either not

synthesized by human astrocytes or produced in very low

amounts by an alternative pathway (Baran and Schwarcz

1990) mediated by KYNase or following a potential uptake

of exogenous 3-HK (Eastman et al. 1992) (Fig. 1).

In accordance with previous studies showing that KAT

immunoreactivity and expression have been detected mostly

in astrocytes in the rat brain (Roberts et al. 1992; Guidetti

et al. 1997), we have shown that human astrocytes express

both KAT-I and KAT-II but with a KAT-II expression

30-fold than that of KAT-I. This enzyme expression is well

correlated with the ability of astrocytes to produce KYNA

from KYN (Speciale and Schwarcz 1990; Guidetti et al.

1997). In accordance with the fact that inhibition of KYN-

OHase leads to increase of KYNA production in mouse

brain (Chiarugi et al. 1996) and previous results from

Speciale and Schwarcz (1990), we showed that astrocytes

produce only a limited quantity of KYNA from KYN

(Fig. 3). Indeed, the ratio KYNA/KYN is 0.0051 (i.e. 195-

fold less KYNA than KYN). This poor production yield is

probably associated with the difference of expression of the

KAT subtypes in human astrocytes (Guidetti et al. 1997).

Nevertheless, we observed a very good correlation between

IFN-g induction of increased expression of KAT-I and

Q6

Q7




UNCORRECTED PROOF

KAT-II, and increased KYNA synthesis in astrocytes. We

con®rmed the hypothesis that the astroglial KP stops at the

KYN step and that this leads chie¯y to the synthesis of

KYNA mainly by KAT-II, in accordance with Schwarcz

et al. (1998) and Speciale and Schwarcz (1990).

Previous immunocytochemical studies have shown that

most of 3-HAO, but not the majority of QPRTase, is

contained in astrocytes (KoÈhler et al. 1988a, b, c; Roberts

et al. 1994). These observations are in accordance with our

molecular results which show strong constitutive 3-HAO

expression and weak QPRTase expression in our astrocyte

cultures. Because of the lack of KYN-OHase expression and

thus 3-HK production, only in the presence of 3-HAA did

3-HAO activity `overtake' QPRTase activity leading to a

limited production of QUIN by astrocytes (Fig. 4b). In our

in vitro conditions, astrocytes were more capable of

catabolizing QUIN than producing it. Moreover, the facts

that: (i) the addition of QUIN increased QPRTase expres-

sion con®rms that the speci®c enzyme for QUIN catabolism

is functional in astrocytes, and (ii) the addition of 3-HAA

increased QPRTase expression, con®rm both that astrocytes

can produce QUIN from 3-HAA even if it is a low amount.

The presence of QUIN in astrocytes is the result of an

imbalance between the activities of the QUIN synthetic and

catabolic enzymes which apparently can only be modi®ed

by addition of 3-HAA leading to saturation of QPRTase

by QUIN.

The exact mechanism of QUIN catabolism by astrocyte

is still not identi®ed. QUIN is probably taken up within the

cell but no speci®c receptor or membranous system for

QUIN has been identi®ed on the astrocyte yet. Moreover,

the fact that several studies have shown that QPRTase is

mainly detected within astrocytes (KoÈhler et al. 1988a, b, c;

Du et al. 1991, 1992, 1993) and that others failed to

detect QUIN within astrocyte (Moffett et al. 1993, 1997),

make it highly likely that QUIN catabolism is intra-

cellular. A less likely possibility, is that QPRTase may be

secreted by astrocytes, leading to an extracellular catabolism

of QUIN. Nonetheless, entry of QUIN and/or release of

QPRTase require the presence of a transport system across

the plasma membrane, the identity of which is still

unknown.

The integration of our results leads us to hypothesize that

KYNase activity is only just enough to bypass the missing

KYN-OHase step via production of minor amounts of

anthranilic acid, which may then lead to 3-HAA production,

and later to minor amounts of QUIN, which are then

catabolized (Fig. 1). This is supported both by previous

studies showing that anthranilic acid can be converted to late

KP metabolites (Bender and McCreanor 1982; Baran and

Schwarcz 1990, 1991; Fujigaki et al. 1998) and secondarily

by the high constitutive expression of 3-HAO by astrocytes.

However, results from Eastman et al. (1992), which showed

that astrocytes are able to take up 3-HK, lead to the

hypothesis that this intermediate KP metabolite may be used

as a substrate for QUIN synthesis by the astrocytes. But

again, there must be a membranous transport system for

3-HK, which is still unknown.

Although, our data relate to fetal astrocytes cultures, we

think that it is highly likely that they re¯ect what is

happening in adult astrocytes. The fetal astrocytes exhibit all

characteristics of mature cells (GFAP1, vimentin1,

proliferation). Moreover, our more recent preliminary

results obtained with primary human adult astrocytes have

shown that the levels of expression of KP enzymes are very

close and well conserved between fetal and adult cells (data

not shown). However, the interest of our human fetal model

is emphasized by the relevance of the rat model for KP

studies. Indeed, the rat has a limited capacity to induce IDO

in brain and thereby a limited capacity for QUIN production

Fig. 8 Model for kynurenine pathway

interactions between astrocytes and

leukocytes during brain in¯ammation.

Q8




UNCORRECTED PROOF

in response to immune activation (Mukhopadhyay et al.

1990; Heyes et al. 1997a, b; Fujigaki et al. 1999).

An interesting aspect of our results is the parallel between

what we have shown and what new drugs have been

developed to modify the KP in preventing or treating brain

damages associated with in¯ammatory diseases, neurode-

generative disorders and stroke. New generations of KYNA

analogues, acting directly on the glutamate receptors, and

the KYN-OHase inhibitors, blocking the synthesis of QUIN

and promoting the formation of KYNA, are both very

promising developments (Stone 2000a, b). The KYN-OHase

inhibitors essentially mimic what the astrocyte does. More-

over, our model may be useful in developing, testing or

validating these new drugs.

Our ®nding of the limited KP expression in human

astrocytes leads to the possibility that astroglial KP may be

involved not only in AIDS dementia complex, but also in the

pathogenesis of a number of other brain diseases (Heyes

et al. 1991, 1992, 1998; Heyes, 1993; Brew et al. 1993,

1996), Huntington's disease (Schwarcz et al. 1988a, b),

Alzheimer's disease (Baran et al. 1999), Down syndrome

(Baran et al. 1996), septicaemia-related encephalopathy

(Heyes and Lackner 1990), toxoplasmosis (Daubener et al.

1993), malaria (Sanni et al. 1998), epilepsy (Du et al. 1993)

and traumatic brain injury (Sinz et al. 1998).

In the absence of macrophages and/or microglia, we

suggest that astrocytes protect neurons by catabolizing any

synthesized QUIN and by producing KYNA, an antagonist

of QUIN (Perkins and Stone 1982). Further, we propose that

during in¯ammation of the brain, activated astrocytes may

`inadvertently' and indirectly contribute to neuronal damage

by producing KYN thus `unwittingly' providing a substrate

for QUIN production by in®ltrating macrophages (Heyes

et al. 1997a) and/or activated resident microglia (Saito et al.

1993a; Heyes et al. 1996) (Fig. 8). In support of this model,

we and colleagues have shown that macrophages and

microglia can metabolize KYN to QUIN (Venkateshan

et al. 1996; Heyes et al. 1997a). Indeed, our results

demonstrate that 12.5 mm of KYN (equivalent to the

concentration of KYN produced by cultured astrocytes

after IFN-g stimulation) is suf®cient to signi®cantly increase

QUIN synthesis by blood macrophages to levels that are

neurotoxic. Moreover, there is evidence that the astrocytic

QPRTase cannot catabolize all the QUIN produced by

in®ltrating macrophages during brain in¯ammation. First,

3-HAO activity is signi®cantly higher than QPRTase within

the brain (Foster et al. 1985, 1986). Secondly, our results

imply that QPRTase levels are easily saturable. For

example, after 48 h, QUIN catabolism in astrocytes

switched to synthesis in the presence of 10 mm (data not

shown) or 100 mm of 3-HAA. Moreover, astrocytes only

degraded < 50% of QUIN added in the culture (Fig. 4). If

this latter catabolic factor is applied to levels of QUIN

produced by macrophages after IFN-g stimulation for 48 h,

the theoretical capacity of astroglial QPRTase to catabolize

QUIN is overcome (Fig. 7). Finally, Nottet et al. (1996)

showed that QUIN production by lipopolysaccharide-activated

macrophages in coculture with primary astrocytes (1 : 1) is

decreased only slightly and the QUIN concentration is still

high enough to be neurotoxic (270 nm).

In conclusion, we have shown that astrocytes lack KYN-

OHase. This could prove neuroprotective in the absence of

in®ltrating macrophages and/or activated resident microglia.

During in¯ammation, however, the in®ltration of activated

macrophages and the activation of microglia could result in

the contribution of astrocytes to neurotoxicity.

Acknowledgements

This work was supported by La Fondation pour la Recherche

Medicale/SIDACTION, Institute LILLY, INSERM, Australian

Brain Foundation, NHMRC (grant 157136), and NSW Health

Department.

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kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection: kynurenine...

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