Am J Physiol Cell Physiol Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 291: C618-C626, 2006. First published May 10, 2006; doi:10.1152/ajpcell.00021.2006
0363-6143/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/4/C618    most recent
00021.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Porcheray, F.
Right arrow Articles by Gras, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Porcheray, F.
Right arrow Articles by Gras, G.

CELLULAR METABOLISM

Glutamate metabolism in HIV-infected macrophages: implications for the CNS

Fabrice Porcheray,1 Cathie Léone,1 Boubekeur Samah,1 Anne-Cécile Rimaniol,2 Nathalie Dereuddre-Bosquet,2 and Gabriel Gras1

1Commissariat à l'Energie Atomique, CEA, DSV/DRM/SNV, Unité Mixte de Recherche E-01 Université Paris-Sud XI, Centre de Recherches du Service de Santé des Armées, IFR13 Institut Paris Sud Cytokines, Laboratoire de Neuro-Immuno-Virologie, Fontenay-aux-Roses; and 2Société de Pharmacologie et d'Immunologie-BIO, Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France

Submitted 19 January 2006 ; accepted in final form 5 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Central nervous system disorders are still a common complication of human immunodeficiency virus (HIV) infection and can lead to dementia and death. They are mostly the consequences of an inflammatory macrophagic activation and relate to glutamate-mediated excitotoxicity. However, recent studies also suggest neuroprotective aspects of macrophage activation through the expression of glutamate transporters and glutamine synthetase. We thus aimed to study whether HIV infection or activation of macrophages could modulate glutamate metabolism in these cells. We assessed the effect of HIV infection on glutamate transporter expression as well as on glutamate uptake by macrophages and showed that glutamate transport was partially decreased in the course of virus replication, whereas excitatory amino acid transporter-2 (EAAT-2) gene expression was dramatically increased. The consequences of HIV infection on glutamine synthetase were also measured and for the first time we show the functional expression of this key enzyme in macrophages. This expression was repressed during virus production. We then quantified EAAT-1 and EAAT-2 gene expression as well as glutamate uptake in differentially activated macrophages and show that the effects of HIV are not directly related to pro- or anti-inflammatory mediators. Finally, this study shows that glutamate transport by macrophages is less affected than what has been described in astrocytes. Macrophages may thus play a role in neuroprotection against glutamate in the infected brain, through their expression of both EAATs and glutamine synthetase. Because glutamate metabolism by activated macrophages is sensitive to both HIV infection and inflammation, it may thus be of potential interest as a therapeutic target in HIV encephalitis.

excitatory amino acid transporter; cystine-glutamate antiporter; glutathione; inflammation; oxidative stress; glutamine synthetase

COGNITIVE IMPAIRMENT remains a common complication of late-stage human immunodeficiency virus type 1 (HIV-1) infection. Before the introduction of highly active antiretroviral therapy (HAART), most virus-infected individuals demonstrated neuropathological abnormalities at autopsy, and a quarter had had neurological manifestations of the disease including cognitive, behavioral, and motor abnormalities, ranging from discrete deficits to HIV-1-associated dementia (HAD) (25, 38). Despite the decrease in the incidence of HAD (27), the greater life expectancy of HAART-treated infected individuals suggests that its prevalence may increase. Moreover, a less severe form of neurological attack, called minor cognitive/motor disorder (MCMD), emerged since the introduction of HAART, that may be more prevalent than dementia (27). Altogether, it appears that neuroAIDS is indeed sensitive to HAART although it remains a significant independent risk factor for AIDS mortality (13).

The best histopathological correlate of HAD is the number of activated mononuclear phagocytes in the central nervous system (CNS) (26), suggesting that HAD is the consequence of indirect mechanisms involving mononuclear phagocytes rather than that of direct virus effect. It is indeed widely accepted that macrophages and microglia play a key role in HIV-induced neurotoxicity, through viral neurotoxin production and more probably through immune activation and subsequent release of neurotoxic factors (for review, see Ref. 33). The importance of macrophages in HIV-associated neuropathogenesis is further underlined by the emergence of specific monocyte subsets in the peripheral blood of patients with HAD, which express specific markers and demonstrate enhanced ability to migrate and secrete neurotoxins (19, 37, 42). Nevertheless, a paradoxical contrast exists between macrophage/microglia activation, that may be set up early in the disease at the asymptomatic pre-AIDS stage (2, 50), and the neuronal apoptosis (1) and loss (14) that occur very late (for review, see Ref. 15).

Glutamate, the major excitatory neurotransmitter (20), is thought to be involved in HIV-induced neurotoxicity (32, 61). A rise in extracellular glutamate concentration leads to neuronal death through hyperactivation of N-methyl-D-aspartate (NMDA) receptors, a mechanism called excitotoxicity. The clearance of extracellular glutamate is ensured by a high-affinity glutamate uptake system called XAG– (48) involving excitatory amino acid transporters (EAAT) (for reviews, see Refs. 10 and 24). Among the five EAAT genes cloned to date, EAAT-1 and EAAT-2 are glia expressed (7, 11, 36, 46) and provide in vivo protection against glutamate toxicity (45). Astrocytes would thus play a key role in neuroprotection against glutamate in the course of HIV infection. However, glutamate uptake by astrocytes is lowered by >60% within 6 h of exposure to either HIV-1 gp120, or Tat, associated with a drop in EAAT-2 expression but not EAAT-1 (17, 35, 62). These findings strongly suggest that during infection astrocytes would not fulfil their protective role. On the other hand our group previously described the expression of functional transporters EAAT-1 and EAAT-2 by human macrophages (43), suggesting that they may also exhibit neuroprotective properties. Another transporter expressed by macrophages is also involved in glutamate homeostasis: the cystine/glutamate antiporter. This heterodimeric transporter includes the CD98 heavy chain and the xCT light chain, which confers substrate specificity (47). It takes up extracellular cystine in exchange for intracellular glutamate through a sodium-independent transport system called xc– (3). The rate of cystine uptake is the limiting factor for the synthesis of the major antioxidant glutathione (GSH). In the presence of high extracellular concentrations of glutamate, the cystine/glutamate antiporter functions in reverse, taking up extracellular glutamate, and leading to cystine starvation and oxidative stress. This glutamate uptake is nevertheless less efficient than that mediated by EAATs (44). Although extracellular glutamate competes with cystine uptake, our group found that it unexpectedly increases both cystine capture and GSH synthesis by macrophages in a dose-dependent manner (44). EAAT expression in macrophages thus leads to glutamate-dependent enhancement of glutathione synthesis by providing intracellular glutamate for direct synthesis of glutathione and also for fueling the intracellular pool of glutamate and trans-stimulating the cystine/glutamate antiporter. EAAT expression by macrophages and microglia has also been described in different rat models and in simian and human AIDS (for review, see Ref. 28). Interestingly, glutamine synthetase is also expressed in cerebral macrophages and microglia in simian AIDS (9), suggesting that these cells may complete the whole glutamate-glutamine cycle in the infected brain. The first aim of this study was thus to evaluate in vitro whether HIV infection affects macrophage capacity to capture and metabolize glutamate, as it does in astrocytes, or whether HIV-infected macrophages could still exhibit a neuroprotective activity by clearing extracellular glutamate and providing substrates for GSH synthesis. In the latter case, macrophages could substitute for astrocytes in glutamate-related functions in the course of HIV infection.

The key role played by inflammation in HIV-related CNS attack also led us to investigate the influence of the cytokinic microenvironment on glutamate uptake and glutamate transporter expression by macrophages. As anti-inflammatory activation of macrophage/microglia may also develop in the infected CNS, when neuronal apoptosis occurs (12, 16, 49) we aimed to evaluate the effect of macrophage activation by either pro- or anti-inflammatory stimuli on glutamate transporter expression and function. Of note, we previously described a bright EAAT expression in perineuronal microglia in HIV infection, that is totally lost when HIV encephalitis and neuronal apoptosis occur (56). In perivascular macrophages and microglial nodules, EAAT expression parallels microgliosis and is maintained in HIV encephalitis cases (56). This particular expression profile suggested that pro- and anti-inflammatory activation pathways in macrophages may differentially modulate the expression of glutamate transporters, further supporting the rationale of the present study. We assessed the effects of the anti-inflammatory cytokines IL-4 and IL-10, which can be released by infiltrating lymphocytes or macrophage/microglia, respectively. The role of PGE2 was measured because this prostaglandin mediates the anti-inflammatory switch of macrophages after phagocytosis of apoptotic cells (16). The pro-inflammatory cytokines tested are major mediators of brain inflammation, TNF-{alpha} and IFN-{gamma}, which can be produced by infiltrating lymphocytes. Dexamethasone and LPS were used as controls for typical anti- and pro-inflammatory activation, respectively.

Using human primary monocyte-derived macrophages (MDM), we hereby show that glutamate transporters are indeed modulated by macrophage activation and infection, but in a complex scheme that is not simply related to pro- and anti-inflammatory features. Nevertheless, this suggests a real, although only partially, protective role for macrophages/microglia against glutamate toxicity in the course of HIV infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human monocyte isolation and differentiation. Monocytes were isolated from healthy seronegative donors by countercurrent elutriation (18). Monocyte preparations were >95% pure, as shown by flow cytometry. One hundred million monocytes were seeded into 75 cm2 culture flasks in DMEM-glutamax medium (Invitrogen, San Diego, CA) supplemented with 10% heat-inactivated (+56°C for 30 min) fetal calf serum (Bio West, Nuaillé, France) and 1% antibiotic mixture (penicillin-streptomycin-neomycin 100x, Invitrogen). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 and 1 ng/ml, respectively; R&D Systems, Minneapolis, MN) were included in the medium from day 0 to day 6. These relative concentrations of cytokines maintained a neutral environment with respect to activation marker expression (HLA-DR, CD163, CD206), which remained similar to that of MDM cultured in medium alone (data not shown). In these conditions, cell survival during differentiation was good. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Blood monocytes adhered to plastic after 1 h, spontaneously detached after 48 h and retained a monocyte-like appearance for 5 days. After 3 days, monocytes were washed with phosphate-buffered saline (PBS), counted using trypan blue, and dispensed into 48-well plates (3 x 105 cells/well) in M-CSF and GM-CSF-supplemented medium. On day 5 and 6, cells were washed and fresh medium lacking M-CSF or GM-CSF was added. On day 7 and 8, mature cells were stimulated with pro- or anti-inflammatory molecules, or infected.

Recombinant cytokines and biologically active substances. Recombinant human IL-4, IL-10, IFN-{gamma}, and TNF-{alpha} were purchased from R&D Systems and used at a concentration of 10 ng/ml for stimulation. Dexamethasone (40 ng/ml) was purchased from Qualimed Laboratories (Paris, France) and PGE2 (10 ng/ml), from Cayman Biochemicals (Ann Arbor, MI). All substances except dexamethasone contained <0.1 ng endotoxin per microgram of product, leading to endotoxin concentrations of <1 pg per ml during the stimulations. Dexamethasone was from a sterile, apyrogenic batch suitable for injection in human. This ensured that the observed results did not arise from endotoxin contamination. LPS (10 ng/ml) was purchased from Sigma (St. Louis, MO). We chose to apply these concentrations for the different stimuli based on those used within the literature to activate macrophages (6, 34, 39, 41, 51, 52).

Real-time quantitative PCR. RNA was extracted using a RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA was treated with 5 units of RNAse-free DNAse (Roche, Mannheim, Germany) for 45 min at room temperature, and DNAse was then inactivated by heating for 5 min at 95°C. RNA was reverse transcribed in optimal conditions, as previously defined (4). PCR reactions were performed in the IQ SybrGreen Supermix (Bio-Rad, Hercules, CA), with PCR cycles, consisting of 15 s at 95°C, 40 s at 60°C, and 20 s at 72°C, in a Icycler apparatus (Bio-Rad). Primers were 5'-TACTCATTCACGCAGTCATCG-3' (sense) and 5'-CCATGTTAATGGTGGCTCCTAC-3' (antisense) for EAAT-1 gene, 5'-CATCTTGGCTCAGAGGAACC-3' (sense) and 5'-AGATGCCAAGCGAAGAAGC-3' (antisense) for EAAT-2 gene, 5'-GACTAATGCCGAGGTCATGC-3' (sense) and 5'-TTGGTGCTGAAGTTGGTATGG-3' (antisense) for GS gene, and 5'-TCGTGGAAGGACTCATGACC-3' (sense), and 5'-TCAGCTCAGGGATGACCTTG-3' (antisense) for GAPDH gene, that was used as endogenous reference to compensate for different RT efficiencies and normalize the variability in the initial quantities of total RNA, so that accurate comparison of gene expression levels could be made between the different samples.

Viruses, infection, and quantification of HIV-1 replication. We used the macrophage-tropic HIV-1 reference strain Ba-L (21). This virus was amplified in vitro using only human PHA-P-activated umbilical blood mononuclear cells. Clarified cell-free umbilical blood mononuclear cell culture supernatants were ultracentrifuged at 360,000 g for 10 min at 4°C just before virus use, to eliminate soluble factors such as cytokines and avoid nonspecific modulations. MDM were infected at a multiplicity of infection (MOI) of 0.1 at day 7–8 of culture. At day 2 post infection (PI), cells were thoroughly washed to remove residual virus. Supernatant was collected twice weekly until day 25 PI, and stored at –20°C to measure the reverse transcriptase (RT) activity by using a commercial kit (RetroSys; Innovagen, Lund, Sweden).

Glutamate uptake. Glutamate uptake was determined using MDM seeded in 48-well plates. The uptake medium was composed of (in mM) 137 NaCl, 0.7 K2HPO4, 1 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.4). We assessed Na+ dependence by replacing the NaCl with 137 mM choline chloride (Sigma). Cells were washed with 1 ml of PBS and incubated for 20 min at 37°C in 200-µl uptake medium, with ionic modifications if necessary. The medium was aspirated and replaced with 100-µl uptake medium (with ionic modifications or inhibitors if necessary) containing L-[2,3-3H]glutamic acid (62 Ci/mmol; Amersham Biosciences, Piscataway, NJ). For glutamate concentrations above 50 mM, [3H]glutamate-specific activity was reduced by a factor of 100 or 200 by diluting in unlabeled glutamate (Sigma). Uptake was stopped after 5 min by removal of medium and by being washed twice with 1 ml of PBS. Cells were then lysed with 130 µl of 100 mM NaOH. The radioactivity of 60 µl of lysate was determined by liquid scintillation counting.

In a series of preliminary experiments, we tested whether protein content quantification in lysates from differently activated macrophages was or was not suitable for assessing MDM number. This method gave the same results as two other cell quantification methods: cell counting with trypan blue exclusion (average number of viable macrophages in one x4 magnification field), and methylthiazolyl diphenyl tetrazolium (MTT) colorimetric assay (data not shown). To correlate glutamate uptake to a fixed number of cells, we thus evaluated the quantity of cells per well by quantifying the lysate protein content.

The protein content of 60 µl of cell lysate was determined with the use of a commercial kit (MicroBC assay, Uptima; Interchim, Montluçon, France). All experiments were performed in triplicate. Glutamate uptake is expressed as a percentage of control MDM uptake activity.

Western blot analysis. MDM from 12 replicate culture wells were lysed at 4°C in 50 µl of lysis buffer [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, and 1% Triton X-100] with protease inhibitor (Roche). Pooled lysate was then centrifugated for 2 min at 12,000 g to remove DNA, followed by a 2-h centrifugation at 4°C and 12,000 g to pellet proteins. The pellet was resuspended in 50 µl of 20 mM Tris pH 8 buffer containing 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin and leupeptin, and 1% Triton X-100. Laemmli buffer containing 100 µM 2-mercaptoethanol was then added, and the samples were boiled for 5 min before being subjected to SDS-PAGE in a 12% acrylamide gel (200 V, 90 min). The separated proteins were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham, Uppsala, Sweden) (100 V, 90 min). Membranes were blocked (3 h, RT) with PBS containing 0.1% Tween 20 and 5% BSA and incubated overnight at 4°C with anti-EAAT-1 or anti-EAAT-2 mouse monoclonal (1:500; Novocastra, Newcastle-upon-Tyne, UK) and anti-actin mouse monoclonal (1:2,000 Calbiochem, San Diego, CA) antibodies. Membranes were then washed three times for 10 min in Tris-buffered saline-Tween 20 (TBS-T) and incubated for 2 h with horseradish peroxidase-conjugated goat anti-mouse antibody (Zymed Laboratories, San Francisco, CA). After two washes in TBS-T and one in TBS, the proteins were subsequently detected using the "ECL Plus" Western Blotting Detection Reagents (Amersham Pharmacia Biotech). Signal intensity on the film was quantified with densitometric analysis software (NIH Image version 1.2; W. Rasband, National Institutes of Health, Bethesda, MD), and the ratio between EAAT and actin signals probed on the same membrane calculated.

Quantification of glutamine synthetase activity. All solutions were maintained at 4°C. Cell cultures were washed with PBS, and the cells were then scraped with the use of a rubber policeman into 75 µl of ultrapure water. The cell lysates were sonicated at 4°C with a 750-W ultrasonic processor (5 pulses of 5 s at maximum power; Vibracell, Sonics, Newtown, CT), aliquoted, and frozen at –20°C.

The measurement of GS activity was performed in 96-well plates, by the addition of 50 µl of cell extract to 75 µl of a reaction mixture containing 0.1 M imidazole-HCl buffer (pH 7.2), 50 mM glutamate, 20 mM adenosine triphosphate, 1 mM beta-mercaptoethanol, 40 mM MgCl2, and 100 mM hydroxylamine hydrochloride (Sigma). After a 60-min incubation at 37°C, the reaction was stopped by the addition of 150 µl/well of stop solution containing 0.37 M FeCl3, 0.2M trichloroacetic acid (Sigma), and 0.67 M HCl. After 30 min at 4°C and 5 min at room temperature, the amount of {gamma}-glutamyl hydroxamate formed in the reaction was read at 540 nm using a Microplate autoreader (model EL311; Biotek Instruments, Winooski, VT) and assessed against a standard curve of known concentrations of {gamma}-glutamyl hydroxamate. Specific activity was normalized against protein content in cell samples (MicroBC assay).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HIV replication upregulates EAAT-2 gene expression in macrophages with poor effect on protein level. EAAT-1, EAAT-2, and xCT gene expression levels were quantified for 3 wk in the course of HIV infection. As shown in Fig. 1A, EAAT-1 and xCT gene expression levels were not modulated whatever the replication level. In contrast, EAAT-2 gene expression was upregulated by >20-fold when HIV replication reached its maximum level. Moreover, EAAT-2 gene upregulation strictly paralleled the level of HIV replication, indicating either that the EAAT-2 gene linearly responds to HIV stimulation or that both events respond to the very same induction cascade. Densitometric analysis of the Western blot suggests that, relative to actin, EAAT-1 protein level decreased with time and was not modulated by infection, while EAAT-2 was stable over time and repeatedly higher in HIV replicating cells. Nevertheless, this effect was of low amplitude (Fig. 1B) and has no statistical value as we did not perform replicate wells to assess intraexperiment signal variation. Contrasting with its gene expression level, EAAT-2 protein level was thus poorly modulated (Fig. 1B) suggesting either a block at the posttranscriptional level or an increased rate of transporter degradation.


Figure 1
View larger version (49K):
[in this window]
[in a new window]
  		Fig. 1. Consequences of human immunodeficiency virus (HIV) replication on the expression of glutamate transporters. Mature macrophages were infected with HIV-1/Ba-L [multiplicity of infection (MOI) = 0.1] and followed up for 21 days thereafter. A: excitatory amino acid transporters (EAAT)-1, EAAT-2 and xCT gene expression levels were measured twice weekly by means of qPCR normalized to GAPDH expression. Results are shown as induction index compared with noninfected controls. The level of HIV replication was assessed by quantifying RT activity in supernatants. Results are expressed as means ± SE of 3 independent experiments. B: EAAT-1 and EAAT-2 protein expression was evaluated by Western blot analysis at days 7, 14, and 21 post infection (PI). Cell extracts were probed using EAAT-1 and EAAT-2 monoclonal antibodies. beta-Actin was used as an internal control of protein content. Densitometric analysis permitted to measure each band's intensity and to calculate the ratio between each EAAT and beta-actin signal (signal ratio). A representative blot of 3 independent experiments is shown.

 
Glutamate uptake by macrophages is decreased in the course of HIV replication. The discrepancy between EAAT-2 expression at the gene and protein levels led us to assess whether glutamate transport is affected or not in HIV-infected MDM. Glutamate uptake was assessed twice weekly over a 24-day post infection period. As shown on Fig. 2, uptake tended to decrease by day 7-10 PI, an effect that was significant at day 14 PI, when a high virus replication occurred. The reduction in glutamate uptake by infected vs. control MDM reached 35%. HIV replication in macrophages thus reduces their capacity to clear extracellular glutamate, but this effect is moderate. Of note, this reduction cannot be attributed to the sodium-independent xc– transport system, which was not affected and accounted for ~10% of the total glutamate uptake (Fig. 2).


Figure 2
View larger version (29K):
[in this window]
[in a new window]
  		Fig. 2. Consequences of HIV infection on glutamate transport by macrophages. Mature macrophages were infected with HIV-1/Ba-L (MOI = 0.1), and glutamate uptake was assessed every 3–4 days all along the replication kinetic in Na+-containing or Na+-free uptake buffer. Transport mediated by the cystine-glutamate antiporter (Na+-independent transport) was measured in Na+-free uptake buffer. Transport mediated by EAATs (Na+-dependent transport) was calculated by subtracting the cystine-glutamate antiporter-mediated uptake from total uptake (performed in Na+-containing buffer). The level of HIV replication was assessed by quantifying RT activity in supernatants. Results are expressed as means ± SE of 3 independent experiments. *P < 0.05 Student's unpaired t-test.

 
Glutamine synthetase is expressed in MDM. Besides EAAT, glutamine synthetase is a critical effector in the trophic glutamate-glutamine cycle. To verify whether the whole cycle could occur in MDM, we tested the expression of the GS gene and the presence of GS activity in MDM. As shown in Fig. 3, both GS gene expression and enzyme activity were detectable throughout the monocyte to macrophage differentiation process.


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Glutamine synthetase is expressed in MDM. Glutamine synthetase gene expression and enzyme activity were assessed during monocyte to macrophage differentiation. A: real time quantitative RT-PCR reveals a constitutive expression of the GS gene, with a tenfold induction at day 1 compared with monocytes, followed by a return to basal level. B: GS activity in cell extracts gradually increases during MDM differentiation. For both RT-PCR and activity, results are expressed as means ± SE of 3 independent experiments.

 
Glutamine synthetase is transcriptionally repressed in the course of HIV replication. Inhibition of glutamate uptake may disturb the dynamic pool of intracellular glutamate, and thus alter substrate availability for the glutamine synthetase. We accordingly assessed whether HIV replication, that decreased EAAT functionality, may also affect GS gene expression and activity levels in macrophages. As shown in Fig. 4, GS gene expression was repressed by ~50% by day 17 PI. GS activity in cell lysates was also repressed in the same proportion, suggesting a transcriptional repression.


Figure 4
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Glutamine synthetase gene expression and activity are repressed in the course of HIV replication. Mature macrophages were infected with HIV-1/Ba-L (MOI = 0.1) at day 0. Glutamine synthetase gene expression and enzyme activity were measured on days 3, 7, 14, 17, and 21 PI. RT activity was similar to the ones shown in Figs. 1 and 2. GS gene expression decreased by ~50% beginning on day 14 PI, whereas the GS activity showed a nonsignificant (NS) increase at day 14 PI, followed by a 50% decrease thereafter. For both RT-PCR and activity, results are expressed as means ± SE of 3 independent experiments. *P < 0.05, Student's unpaired t-test.

 
Pro- and anti-inflammatory activation modulate the expression of glutamate transporters and glutamate uptake. To assess the role of the inflammatory environment on both the expression of transporters and on glutamate uptake, mature macrophages were treated for 3 days with different pro- and anti-inflammatory agents. Then, real-time PCR for EAAT-1, EAAT-2, and xCT genes, as well as glutamate uptake, were performed. EAAT-1 gene expression was on the whole poorly modulated by the tested stimuli (Fig. 5), except for dexamethasone that increased it 5-fold. Pro-inflammatory cytokines and LPS induced a weak 2-fold upregulation of EAAT-1 that did not test significant (P = 0.27; 0.12; 0.12 for IFN-{gamma}; TNF-{alpha} and LPS, respectively, t-test).


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5. Effect of pro- and anti-inflammatory stimuli on transporter gene expression and glutamate uptake. Mature macrophages were treated for 3 days with 10 ng/ml [except for 40 ng/ml dexamethasone (Dex)] of each molecule. Gene expression and glutamate uptake capacity were assessed on replicate wells of the same cells. Data are expressed as percentage of untreated cells and are means ± SE of 3 independent experiments. *P < 0.05, Student's unpaired t-test.

 
EAAT-2 was much more responsive to inflammation-related stimuli than EAAT-1 (Fig. 5). PGE2, which does not polarize cells towards a clear pro- or anti-inflammatory phenotype (41), as well as LPS, upregulated EAAT-2 mRNA by about 20- and 10-fold, respectively. Other stimulations had virtually no effect on EAAT-2 gene expression, suggesting that its increase observed during HIV replication did not arise from a shift toward a specific (anti) inflammatory pattern. Like EAAT-2, xCT expression was inducible by LPS, but also by TNF-{alpha}, and to a lesser extent by PGE2. This suggests that EAAT-2 and xCT genes act in concert for an adaptive response to PGE2, TNF-{alpha}, and LPS.

IL-4 decreased the capacity of MDM to take up extracellular glutamate by ~50%, although it did not test significant. IL-4 had no effect on EAAT-1 and EAAT-2 gene expression but significantly increased xCT expression. This overexpression of xCT is probably not relevant for glutamate transport because the cystine-glutamate antiporter accounts for <10% of the total glutamate uptake (see Fig. 2). In contrast, TNF-{alpha} and LPS led to an increased uptake (Fig. 5), whereas dexamethasone and PGE2 induced EAAT-1 and EAAT-2 gene expression, respectively, but without a significant increase in glutamate uptake, possibly due to the intricate regulation of EAAT at the posttranscriptional level (for review, see Ref. 10).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The role of macrophages in CNS infection by HIV has so far been restricted to pro-inflammatory features with deleterious consequences for neurons through the release of pro-inflammatory cytokines, viral proteins, and other neurotoxins, including glutamate (for review, see Ref. 33). Although these features are undeniable and well documented, the demonstration by our group of efficient glutamate transport by human macrophages (43) led us to investigate whether macrophage activation could also entail protective aspects with regard to glutamate metabolism. In this report, we first augment our previous findings and demonstrate that MDM also express glutamine synthetase at the gene and enzymatic activity levels. Together with EAAT, GS is a critical effector of the trophic and protective glutamate-glutamine cycle. Nevertheless, whether this coexpression of EAAT and GS in activated macrophages leads to the provision of the trophic amino acid glutamine to neighboring neurones remains to be determined, as we assessed neither glutamine secretion nor the expression of system N or ASCT2 transporters that mediate glutamine efflux from astrocytes (5, 8, 29). In this study, we showed that glutamate transport is less repressed by HIV in macrophages than what was described by others in astrocytes. Indeed, Wang et al. (58) observed a >60% reduction in glutamate uptake by HIV-infected or gp120-stimulated astrocytes. On the other hand, macrophage uptake capacity was only partially lowered throughout a 25-day infection kinetic study, and this decrease appeared only when high replication occurred, suggesting that virus contact per se may not allow such an effect. This relative insensitivity of macrophages to HIV replication suggests that these cells may, at least partially, fill in the defect of astrocytic glutamate uptake in the infected brain.

At the gene expression level, HIV replication in macrophages induced a striking increase in EAAT-2 gene expression. The physiological significance of this EAAT-2 mRNA overexpression observed in the course of infection remains to be elucidated, bearing in mind that it was followed by a poor increase in protein content, and even a diminished transporter function. The dissociation between gene expression and transporter function may relate to posttranscriptional and/or transporter trafficking events, as already shown in astrocytes where, for example, AMPA-kainate receptor activation upregulates GLAST (the murine EAAT-1) activity without affecting its mRNA level (22). Interestingly, the presence of functional AMPA-kainate receptors has also been demonstrated on brain macrophages, suggesting that they may also respond to extracellular glutamate (40). We thus tested whether infected macrophages may regulate their glutamate uptake capacity in response to an excess of extracellular glutamate. To this end, we assayed glutamate uptake 12 h after treating both uninfected and infected macrophages with 10 mM glutamate. This stimulation had no effect (data not shown), suggesting that other regulatory pathways are responsible for the gap between EAAT-2 gene expression, its protein content, and its function. Vanoni et al. (57) showed in a mouse model of amyotrophic lateral sclerosis that the murine counterpart of EAAT-2, GLT-1, exhibits increased internalization and degradation through aberrant oxidative stress, whereas the other glutamate transporters are not affected. If active in our system, such a mechanism would explain the discrepancy between EAAT-2 gene expression, its protein amount, and its function. The establishment of an oxidative stress in HIV replicating macrophages is strongly supported by different studies. First, Yeh et al. (59) reported that HIV-1-infected MDM produce significantly less cysteine than uninfected ones, following TNF-{alpha} stimulation. This indicates that HIV replication consumes cysteine. Cysteine is the limiting amino acid precursor for GSH synthesis, and its decreased availability in infected MDM suggests that HIV replication may alter the GSH synthesis pathway. Concordant with this idea, Mialocq et al. (38a) showed that HIV replication is indeed associated with a significant decrease in intracellular GSH concentration, as well as transcriptional modulation of enzymes involved in the regulation of intracellular GSH levels, leading to oxidative stress. The involvement of oxidative stress in EAAT-2 expression is still a matter of debate (53), but one can suggest that HIV replication in MDM might account for a decrease in EAAT-2 expression at the cell membrane, as suggested by amyotrophic lateral sclerosis data (57). In this hypothesis, the strong overexpression of EAAT-2 mRNA that we observed during HIV infection, leading to slight increase in protein content, might be a compensatory mechanism to counterbalance this degradation. Pulse-chase experiments to measure EAAT-2 protein turnover would answer this question. In addition to transporter degradation, mediators of oxidative stress also inhibit EAAT function through a sulfhydryl sensor based mechanism (54). Finally, regardless of whether it acts through the degradation of transporter protein or through the regulation of its function, the HIV-induced oxidative stress is likely to be responsible for the observed repression of glutamate uptake.

Another mechanism that may account for the overexpression of the EAAT-2 gene involves the cystine/glutamate antiporter. Because the need for cysteine increases during HIV replication, and because cystine is the most abundant extracellular precursor of cysteine, the xc– system would act to replenish the cysteine pool. By uptaking cystine, this system releases glutamate, a consequence of which is a drop in the glutamate gradient that would stimulate the EAAT system via an overexpression of EAAT-2. The sodium-independent cystine/glutamate antiporter transport capacity was not modified throughout HIV infection, and its role in cystine uptake would thus not be altered. Nevertheless, it is known from previous studies (44) that intricate interactions between the xc– and XAG– transport systems regulate GSH synthesis in MDM. The observed perturbations of the EAAT (XAG–) expression and function may thus arise from normal transport systems interaction rather than from a defect of the cystine/glutamate antiporter (xc–). The interactions between these transport systems, and their perturbation by HIV infection and oxidative stress are schematized in Fig. 6.


Figure 6
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 6. Proposed mechanism for glutamate uptake and glutamine synthetase inhibition in HIV-replicating macrophages. 1) In normal conditions, XAG– and Xc– transport systems cooperate to regulate glutathione (GSH) synthesis. The xc– antiporter takes up cystine, the limiting extracellular precursor for GSH, in exchange for glutamate thus depleting the intracellular pool of glutamate. In turn, XAG– transport restores the glutamate concentration gradient, trans-stimulating cystine uptake and fuelling the intracellular pool of glutamate. Glutamate is either inserted into GSH, metabolized into the trophic aminoacid glutamine, or secreted during cystine uptake. 2) HIV replication is cysteine consuming and disturbs GSH metabolism, leading to glutathione depletion and oxidative stress. 3) Oxidative stress directly lowers XAG– transport capacity. This alteration of EAAT function and the demand for cysteine lead to a decreased glutamate concentration gradient and glutamate availability, further contributing to GSH depletion and oxidative stress. 4) The repression of glutamine synthetase may be a consequence of lowered glutamate availability.

 
Whatever the reason for the drop in glutamate transport, one important consequence of this drop may be the decrease in glutamine synthetase expression. This 50% decrease occurred at both the transcriptional and enzyme activity levels. It seemingly corresponds to a transcriptional repression in response to a disturbed glutamate pool. From this point of view, we must keep in mind that the glutamate pool, although abundant, is dynamic and can easily be deprived (for review, see Ref. 28).

Our study, as well as others (23, 30), emphasizes the preponderant role of EAAT-2 in glutamate transport adaptation to changes in the external conditions. Indeed, EAAT-1 was not modulated at the mRNA or the protein level. As this transporter may account for ~70% of glutamate uptake by MDM (43), its refractory state to external stimuli may contribute to the moderate modulation of glutamate uptake we observed.

Beside the effects of HIV replication, our data also clearly show that EAAT gene expression and function can be modulated by either pro- or anti-inflammatory stimuli. Again, EAAT-2 was the most responsive to stimulations, suggesting that it may fulfil a predominant role in maintaining glutamate homeostasis through rapid responses. EAAT-mediated glutamate uptake by MDM is not simply linked to inflammatory activation because both pro- and anti-inflammatory molecules can increase it, suggesting that inflammation per se is not the key regulator for these transporters. Although activation can induce high EAAT overexpression at the mRNA level, modulation of glutamate uptake level in macrophages is of lower magnitude, suggesting an intricate control through finely tuned posttranscriptional regulation of the different transporters. However, modulation of glutamate transport grossly fits gene expression patterns. Moreover, the amplitude of the variations we observed are in the same range as those induced by other recognized EAAT regulation mediators such as polyunsaturated fatty acids (55, 60). This suggests that the observed effects may indeed have relevant functional consequences.

PGE2, which production by macrophage/microglia is markedly increased after phagocytosis of apoptotic cells (12, 16), strongly upregulated EAAT-2 expression and slightly increased glutamate uptake in our conditions. This feature may be a component of the protective action of PGE2 in different neurological diseases (12). Likewise, LPS also induced MDM to overexpress EAAT-2 mRNA, and increased glutamate transport. Such induction may relate to the need to maintain the glutamate concentration gradient in conditions where microbicidal functions of macrophages entail a demand for GSH synthesis, and thus cystine uptake and glutamate secretion (44). These data suggest that extracellular glutamate concentration may be strictly regulated in the vicinity of macrophages through modulation of EAAT expression and function.

Taken together, our data suggest that HIV replication in MDM disturbs the balance between glutamate uptake and secretion, and leads to EAAT-2 gene induction. The signification of this EAAT-2 gene induction still requires further investigation to be fully understood. This may nevertheless participate in the important uncoupling that is observed between macrophage/microglia activation throughout the infected brain, with its well documented neurotoxic consequences, and neuronal apoptosis that occurs late in this process. Different effectors of macrophage activation seemingly also modulate these equilibriums. Nevertheless, the fact that only TNF-{alpha} and LPS are able to significantly enhance glutamate uptake supports the idea that the inflammation that occurs within the CNS during HIV infection, that is mainly mediated by TNF-{alpha} (for review, see Ref. 33), plays a major role in glutamate-mediated neurotoxicity. Indeed, TNF-{alpha} induces the production and release of nitric oxide (31) that generate an oxidative stress likely compensated by an increase in glutathione synthesis via cystine uptake and glutamate release through the cystine/glutamate antiporter. Thus compensatory mechanisms involving EAAT must also be set up in response to inflammatory mediators and not only in response to HIV replication. The effect of LPS on glutamate uptake leads to similar mechanisms during bacterial infection.

In conclusion, our results strongly support the hypothesis that modulation of glutamate transport and glutamine synthetase in activated macrophages responds to the natural functions of macrophages that necessitate adapting GSH metabolism during defense against microorganisms. On the other hand, this protective profile that includes EAAT and GS expression in macrophages is altered upon HIV replication, although this effect is probably less important than the HIV-induced depression of the same functions in astrocytes. The fine interactions between, on the one hand, the protective glutamate-glutamine cycle and the GSH metabolic pathways in macrophages/microglia and astrocytes, and, on the other hand, the level of HIV replication and the subsequent inflammatory activation features may be critical to the local and temporal outcome of the neuron survival/death decision.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS) and Ensemble Contre le SIDA (Sidaction), and recurrent funds of the Commissariat à l'Energie Atomique. F. Porcheray, B. Samah, and C. Léone hold doctoral fellowships from the ANRS. F. Porcheray was a recipient of a fellowship from the Fondation pour la Recherche Médicale.


   ACKNOWLEDGMENTS
 
We thank Dr. Ann Mullally for kindly correcting the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Gras, Laboratoire de Neuro-Immuno-Virologie, Commissariat à l'Energie Atomique, DSV/DRM/SNV, 18 route du Panorama, F92265 Fontenay-aux-Roses cedex, France (e-mail: gabriel.gras{at}cea.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Adle-Biassette H, Levy Y, Colombel M, Poron F, Natchev S, Keohane C, and Gray F. Neuronal apoptosis in HIV infection in adults. Neuropathol Appl Neurobiol 21: 218–227, 1995.[Web of Science][Medline]

2. An SF, Ciardi A, Giometto B, Scaravilli T, Gray F, and Scaravilli F. Investigation on the expression of major histocompatibility complex class II and cytokines and detection of HIV-1 DNA within brains of asymptomatic and symptomatic HIV-1-positive patients. Acta Neuropathol (Berl) 91: 494–503, 1996.[CrossRef][Medline]

3. Bannai S. Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem 261: 2256–2263, 1986.[Abstract/Free Full Text]

4. Benveniste O, Vaslin B, Villinger F, Le Grand R, Ansari AA, and Dormont D. Cytokine mRNA levels in unmanipulated (ex vivo) and in vitro stimulated monkey PBMCs using a semi-quantitative RT-PCR and high sensitivity fluorescence-based detection strategy. Cytokine 8: 32–41, 1996.[CrossRef][Web of Science][Medline]

5. Broer A, Brookes N, Ganapathy V, Dimmer KS, Wagner CA, Lang F, and Broer S. The astroglial ASCT2 amino acid transporter as a mediator of glutamine efflux. J Neurochem 73: 2184–2194, 1999.[Web of Science][Medline]

6. Buechler C, Ritter M, Orso E, Langmann T, Klucken J, and Schmitz G. Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli. J Leukoc Biol 67: 97–103, 2000.[Abstract]

7. Chaudhry FA, Lehre KP, van Lookeren Campagne M, Ottersen OP, Danbolt NC, and Storm-Mathisen J. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Neuron 15: 711–720, 1995.[CrossRef][Web of Science][Medline]

8. Chaudhry FA, Reimer RJ, Krizaj D, Barber D, Storm-Mathisen J, Copenhagen DR, and Edwards RH. Molecular analysis of system N suggests novel physiological roles in nitrogen metabolism and synaptic transmission. Cell 99: 769–780, 1999.[CrossRef][Web of Science][Medline]

9. Chretien F, Vallat-Decouvelaere AV, Bossuet C, Rimaniol AC, Le Grand R, Le Pavec G, Creminon C, Dormont D, Gray F, and Gras G. Expression of excitatory amino acid transporter-2 (EAAT-2) and glutamine synthetase (GS) in brain macrophages and microglia of SIVmac251-infected macaques. Neuropathol Appl Neurobiol 28: 410–417, 2002.[CrossRef][Web of Science][Medline]

10. Danbolt NC. Glutamate uptake. Prog Neurobiol 65: 1–105, 2001.[CrossRef][Web of Science][Medline]

11. Danbolt NC, Storm-Mathisen J, and Kanner BI. An [Na+ + K+] coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience 51: 295–310, 1992.[CrossRef][Web of Science][Medline]

12. De Simone R, Ajmone-Cat MA, and Minghetti L. Atypical antiinflammatory activation of microglia induced by apoptotic neurons: possible role of phosphatidylserine-phosphatidylserine receptor interaction. Mol Neurobiol 29: 197–212, 2004.[CrossRef][Web of Science][Medline]

13. Ellis RJ, Deutsch R, Heaton RK, Marcotte TD, McCutchan JA, Nelson JA, Abramson I, Thal LJ, Atkinson JH, Wallace MR, and Grant I. Neurocognitive impairment is an independent risk factor for death in HIV infection. San Diego HIV Neurobehavioral Research Center Group. Arch Neurol 54: 416–424, 1997.[Abstract/Free Full Text]

14. Everall I, Gray F, Barnes H, Durigon M, Luthert P, and Lantos P. Neuronal loss in symptom-free HIV infection. Lancet 340: 1413, 1992.[CrossRef][Web of Science][Medline]

15. Everall I, Luthert P, and Lantos P. A review of neuronal damage in human immunodeficiency virus infection: its assessment, possible mechanism and relationship to dementia. J Neuropathol Exp Neurol 52: 561–566, 1993.[Web of Science][Medline]

16. Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, and Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest 101: 890–898, 1998.[Web of Science][Medline]

17. Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, Belladonna ML, Fioretti MC, Alegre ML, and Puccetti P. Modulation of tryptophan catabolism by regulatory T cells. Nat Immun 4: 1206–1212, 2003.

18. Figdor CG, Leemans JM, Bont WS, and de Vries JE. Theory and practice of centrifugal elutriation (CE). Factors influencing the separation of human blood cells. Cell Biophys 5: 105–118, 1983.[Web of Science][Medline]

19. Fischer-Smith T, Croul S, Sverstiuk AE, Capini C, L'Heureux D, Regulier EG, Richardson MW, Amini S, Morgello S, Khalili K, and Rappaport J. CNS invasion by CD14+/CD16+ peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection. J Neurovirol 7: 528–541., 2001.[CrossRef][Web of Science][Medline]

20. Fonnum F. Glutamate: a neurotransmitter in mammalian brain. J Neurochem 42: 1–11, 1984.[Web of Science][Medline]

21. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, and Popovic M. The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233: 215–219, 1986.[Abstract/Free Full Text]

22. Gegelashvili G, Civenni G, Racagni G, Danbolt NC, Schousboe I, and Schousboe A. Glutamate receptor agonists up-regulate glutamate transporter GLAST in astrocytes. Neuroreport 8: 261–265, 1996.[Web of Science][Medline]

23. Gegelashvili G, Danbolt NC, and Schousboe A. Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J Neurochem 69: 2612–2615, 1997.[Web of Science][Medline]

24. Gegelashvili G and Schousboe A. High affinity glutamate transporters: regulation of expression and activity. Mol Pharmacol 52: 6–15, 1997.[Abstract/Free Full Text]

25. Gendelman HE, Persidsky Y, Ghorpade A, Limoges J, Stins M, Fiala M, and Morrisett R. The neuropathogenesis of the AIDS dementia complex. AIDS 11: S35–45, 1997.

26. Glass JD, Fedor H, Wesselingh SL, and McArthur JC. Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia. Ann Neurol 38: 755–762, 1995.[CrossRef][Web of Science][Medline]

27. Gonzalez-Scarano F and Martin-Garcia J. The neuropathogenesis of AIDS. Nat Rev Immunol 5: 69–81, 2005.[CrossRef][Web of Science][Medline]

28. Gras G, Chretien F, Vallat-Decouvelaere AV, Le Pavec G, Porcheray F, Bossuet C, Leone C, Mialocq P, Dereuddre-Bosquet N, Clayette P, Le Grand R, Creminon C, Dormont D, Rimaniol AC, and Gray F. Regulated expression of sodium-dependent glutamate transporters and synthetase: a neuroprotective role for activated microglia and macrophages in HIV infection? Brain Pathol 13: 211–222, 2003.[Web of Science][Medline]

29. Gu S, Roderick HL, Camacho P, and Jiang JX. Identification and characterization of an amino acid transporter expressed differentially in liver. Proc Natl Acad Sci USA 97: 3230–3235, 2000.[Abstract/Free Full Text]

30. Guo H, Lai L, Butchbach ME, Stockinger MP, Shan X, Bishop GA, and Lin CL. Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice. Hum Mol Genet 12: 2519–2532, 2003.[Abstract/Free Full Text]

31. Jiang H, Stewart CA, and Leu RW. Tumor-derived factor synergizes with IFN-gamma and LPS, IL-2 or TNF-alpha to promote macrophage synthesis of TNF-alpha and TNF receptors for autocrine induction of nitric oxide synthase and enhanced nitric oxide-mediated tumor cytotoxicity. Immunobiology 192: 321–342, 1995.[Web of Science][Medline]

32. Jiang ZG, Piggee C, Heyes MP, Murphy C, Quearry B, Bauer M, Zheng J, Gendelman HE, and Markey SP. Glutamate is a mediator of neurotoxicity in secretions of activated HIV-1-infected macrophages. J Neuroimmunol 117: 97–107, 2001.[CrossRef][Web of Science][Medline]

33. Kaul M, Garden GA, and Lipton SA. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410: 988–994, 2001.[CrossRef][Medline]

34. Kodelja V, Muller C, Politz O, Hakij N, Orfanos CE, and Goerdt S. Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern. J Immunol 160: 1411–1418, 1998.[Abstract/Free Full Text]

35. Kort JJ. Impairment of excitatory amino acid transport in astroglial cells infected with the human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 14: 1329–1339, 1998.[Web of Science][Medline]

36. Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, and Danbolt NC. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci 15: 1835–1853, 1995.[Abstract]

37. Luo X, Carlson KA, Wojna V, Mayo R, Biskup TM, Stoner J, Anderson J, Gendelman HE, and Melendez LM. Macrophage proteomic fingerprinting predicts HIV-1-associated cognitive impairment. Neurology 60: 1931–1937, 2003.[Abstract/Free Full Text]

38. Masliah E, DeTeresa RM, Mallory ME, and Hansen LA. Changes in pathological findings at autopsy in AIDS cases for the last 15 years. AIDS 14: 69–74, 2000.[CrossRef][Web of Science][Medline]

38a. Mialocq P, Rimaniol AC, Dereuddre-Bosquet N, Oiry J, Puy JY, Gras G, Imbach JL, Dormont D, and Clayette P. Dysregulation in the GSH system as a consequence of HIV-1 replication in human macrophages and pro-glutathione compounds as alternative therapeutical strategy in neuroAIDS. 5th International Workshop on HIV, Cells of Macrophage/Dendritic Lineage and Other Reservoirs. Rome, October 13–15, 2002; Poster.

39. Montaner LJ, da Silva RP, Sun J, Sutterwala S, Hollinshead M, Vaux D, and Gordon S. Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10. J Immunol 162: 4606–4613., 1999.[Abstract/Free Full Text]

40. Noda M, Nakanishi H, Nabekura J, and Akaike N. AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia. J Neurosci 20: 251–258, 2000.[Abstract/Free Full Text]

41. Porcheray F, Viaud S, Rimaniol AC, Leone C, Samah B, Dereuddre-Bosquet N, Dormont D, and Gras G. Macrophage activation switching: an asset for the resolution of inflammation. Clin Exp Immunol 142: 481–489, 2005.[Web of Science][Medline]

42. Pulliam L, Gascon R, Stubblebine M, McGuire D, and McGrath MS. Unique monocyte subset in patients with AIDS dementia. Lancet 349: 692–695, 1997.[CrossRef][Web of Science][Medline]

43. Rimaniol AC, Haik S, Martin M, Le Grand R, Boussin FD, Dereuddre-Bosquet N, Gras G, and Dormont D. Na+-dependent high-affinity glutamate transport in macrophages. J Immunol 164: 5430–5438, 2000.[Abstract/Free Full Text]

44. Rimaniol AC, Mialocq P, Clayette P, Dormont D, and Gras G. Role of glutamate transporters in the regulation of glutathione levels in human macrophages. Am J Physiol Cell Physiol 281: C1964–C1970, 2001.[Abstract/Free Full Text]

45. Rothstein JD. Excitotoxicity hypothesis. Neurology 47: S19–26, 1996.[Web of Science][Medline]

46. Rothstein JD, Dykes-Hoberg M, Pardo CA, Bristol LA, Jin L, Kuncl RW, Kanai Y, Hediger MA, Wang Y, Schielke JP, and Welty DF. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16: 675–686, 1996.[CrossRef][Web of Science][Medline]

47. Sato H, Tamba M, Ishii T, and Bannai S. Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem 274: 11455–11458, 1999.[Abstract/Free Full Text]

48. Schousboe A. Transport and metabolism of glutamate and GABA in neurons are glial cells. Int Rev Neurobiol 22: 1–45, 1981.[Medline]

49. Shi B, De Girolami U, He J, Wang S, Lorenzo A, Busciglio J, and Gabuzda D. Apoptosis induced by HIV-1 infection of the central nervous system. J Clin Invest 98: 1979–1990, 1996.[Web of Science][Medline]

50. Sinclair E, Gray F, Ciardi A, and Scaravilli F. Immunohistochemical changes and PCR detection of HIV provirus DNA in brains of asymptomatic HIV-positive patients. J Neuropathol Exp Neurol 53: 43–50, 1994.[Web of Science][Medline]

51. Stein M, Keshav S, Harris N, and Gordon S. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med 176: 287–292, 1992.[Abstract/Free Full Text]

52. Sulahian TH, Hogger P, Wahner AE, Wardwell K, Goulding NJ, Sorg C, Droste A, Stehling M, Wallace PK, Morganelli PM, and Guyre PM. Human monocytes express CD163, which is upregulated by IL-10 and identical to p155. Cytokine 12: 1312–1321, 2000.[CrossRef][Web of Science][Medline]

53. Tortarolo M, Crossthwaite AJ, Conforti L, Spencer JP, Williams RJ, Bendotti C, and Rattray M. Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress. J Neurochem 88: 481–493, 2004.[Web of Science][Medline]

54. Trotti D, Rizzini BL, Rossi D, Haugeto O, Racagni G, Danbolt NC, and Volterra A. Neuronal and glial glutamate transporters possess an SH-based redox regulatory mechanism. Eur J Neurosci 9: 1236–1243, 1997.[CrossRef][Web of Science][Medline]

55. Trotti D, Volterra A, Lehre KP, Rossi D, Gjesdal O, Racagni G, and Danbolt NC. Arachidonic acid inhibits a purified and reconstituted glutamate transporter directly from the water phase and not via the phospholipid membrane. J Biol Chem 270: 9890–9895, 1995.[Abstract/Free Full Text]

56. Vallat-Decouvelaere AV, Chretien F, Gras G, Le Pavec G, Dormont D, and Gray F. Expression of excitatory amino acid transporter-1 in brain macrophages and microglia of HIV-infected patients. A neuroprotective role for activated microglia? J Neuropathol Exp Neurol 62: 475–485, 2003.[Web of Science][Medline]

57. Vanoni C, Massari S, Losa M, Carrega P, Perego C, Conforti L, and Pietrini G. Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS). J Cell Sci 117: 5417–5426, 2004.[Abstract/Free Full Text]

58. Wang Z, Pekarskaya O, Bencheikh M, Chao W, Gelbard HA, Ghorpade A, Rothstein JD, and Volsky DJ. Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120. Virology 312: 60–73, 2003.[CrossRef][Web of Science][Medline]

59. Yeh MW, Kaul M, Zheng J, Nottet HS, Thylin M, Gendelman HE, and Lipton SA. Cytokine-stimulated, but not HIV-infected, human monocyte-derived macrophages produce neurotoxic levels of l-cysteine. J Immunol 164: 4265–4270, 2000.[Abstract/Free Full Text]

60. Zerangue N, Arriza JL, Amara SG, and Kavanaugh MP. Differential modulation of human glutamate transporter subtypes by arachidonic acid. J Biol Chem 270: 6433–6435, 1995.[Abstract/Free Full Text]

61. Zhao J, Lopez AL, Erichsen D, Herek S, Cotter RL, Curthoys NP, and Zheng J. Mitochondrial glutaminase enhances extracellular glutamate production in HIV-1-infected macrophages: linkage to HIV-1 associated dementia. J Neurochem 88: 169–180, 2004.[Web of Science][Medline]

62. Zhou BY, Liu Y, Oh Kim B, Xiao Y, and He J. Astrocyte activation and dysfunction and neuron death by HIV-1 Tat expression in astrocytes. Mol Cell Neurosci 27: 296–305, 2004.[Web of Science][Medline]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/4/C618    most recent
00021.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Porcheray, F.
Right arrow Articles by Gras, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Porcheray, F.
Right arrow Articles by Gras, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.