Transport in Macrophages -Dependent High-Affinity Glutamate

Na؉-Dependent High-Afﬁnity Glutamate Transport in Macrophages1

Anne-Ce´cile Rimaniol,* Ste´phane Haı¨k,* Marc Martin,* Roger Le Grand,* Franc¸ois Dominique Boussin,† Nathalie Dereuddre-Bosquet,*‡ Gabriel Gras,2* and Dominique Dormont*

Excessive accumulation of glutamate in the CNS leads to excitotoxic neuronal damage. However, glutamate clearance is essentially mediated by astrocytes through Na؉-dependent high-affinity glutamate transporters (excitatory amino acid transporters (EAATs)). Nevertheless, EAAT function was recently shown to be developmentally restricted in astrocytes and undetectable in mature astrocytes. This suggests a need for other cell types for clearing glutamate in the brain. As blood monocytes infiltrate the CNS in traumatic or inflammatory conditions, we addressed the question of whether macrophages expressed EAATs and were involved in glutamate clearance. We found that macrophages derived from human blood monocytes express both the cystine/ glutamate antiporter and EAATs. Kinetic parameters were similar to those determined for neonatal astrocytes and embryonic neurons. Freshly sorted tissue macrophages did not possess EAATs, whereas cultured human spleen macrophages and cultured neonatal murine microglia did. Moreover, blood monocytes did not transport glutamate, but their stimulation with TNF-␣ led to functional transport. This suggests that the acquisition of these transporters by macrophages could be under the control of inflammatory molecules. Also, monocyte-derived macrophages overcame glutamate toxicity in neuron cultures by clearing this molecule. This suggests that brain-infiltrated macrophages and resident microglia may acquire EAATs and, along with astrocytes, regulate extracellular glutamate concentration. Moreover, we showed that EAATs are involved in the regulation of glutathione synthesis by providing intracellular glutamate. These observations thus offer new insight into the role of macrophages in excitotoxicity and in their response to oxidative stress. The Journal of Immunology, 2000, 164: 5430–5438.

n the CNS, glutamate plays a major role as a neurotransmit- similar affinities and couples the electrochemical gradient of three ter. At high extracellular concentrations, glutamate is also a cotransported sodium ions and one countertransported potassium I powerful neurotoxin capable of inducing severe excitotoxic ion with that of the amino acids (12). EAAT1 and EAAT2 were damage to neurons (1). primarily observed in astrocytes, and EAAT3 is a neuronal trans- Extracellular glutamate concentration is regulated by transporter porter with a somatodendritic location (13). EAAT gene knockout proteins primarily observed in neurons and astrocytes. These trans- showed that the astroglial transporters EAAT1 and EAAT2 are porters are essential for ensuring a high signal-to-noise ratio and involved in protection against excitotoxicity by clearing extracel- preventing neuronal damage (for review, see Ref. 2). Many neu- lular glutamate, whereas EAAT3 is not (14, 15). Nevertheless, rological diseases may be associated with glutamate transport fail- Stanimirovic et al. (16) recently showed that EAAT function is ure (3–5). Five subtypes of high-affinity glutamate transporters developmentally restricted in cultured astrocytes. Indeed, embry- (excitatory amino acid transporters 1–5 (EAAT1–5)3) have been onic and early postnatal astrocytes (P0) express high EAAT levels cloned from mammalian tissues (6–11). They form a new family in vitro, but glutamate uptake drops in P10–P21 astrocytes and of molecules, with 50–55% amino acid sequence identities. This becomes undetectable in P50 astrocytes (16). This finding suggests Ϫ transport system, XAG , transports L-Asp, D-Asp, and L-Glu with that glutamate clearance in mature brain would need the contribu- tion of other cell types. During brain injury, the CNS parenchyma is open to infiltration *Service de Neurovirologie CEA, DSV/DRM, Centre de Recherches du Service de Sante´ des Armeés, IPSC, Commissariat a` l’Energie Atomique, Fontenay-aux-Roses, by blood cells, resulting in a mixed population of inflammatory France; †Laboratoire de Radiopathologie, DSV/DRR, Commissariat a` l’Energie cells in the damaged tissue. Many studies have shown that most of ‡ Atomique, Fontenay-aux-Roses, France; and Socie´te de Pharmacologie et Immuno- the macrophages present in brain lesions originate from blood logic Bio., Massy, France monocytes (17). Brain macrophages are important effectors of the Received for publication October 25, 1999. Accepted for publication March 7, 2000. local immune response, although they are thought to contribute to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance neurotoxicity by producing inflammatory cytokines, quinolic acid, with 18 U.S.C. Section 1734 solely to indicate this fact. and also glutamate (18–21). Glutamate release by stimulated mac- 1 This work was supported in part by grants from the Agence Nationale de Recherches rophages and microglia is mediated by a cystine/glutamate trans- sur le SIDA (ANRS) and Sidaction. A.-C.R. is a recipient of a fellowship from the port system other than EAATs. This system, X Ϫ,isaNaϩ-inde- ANRS. c pendent anionic amino acid transport present in numerous cell 2 Address correspondence and reprint requests to Dr. Gabriel Gras, Service de Neu- rovirologie, DSV/DRM, Commissariat a` l’Energie Atomique, BP 6, 60–68 avenue de types both in the CNS and the periphery. Generally, cystine is la division Leclerc, 92265 Fontenay-aux-Roses, France. E-mail address: gras@dsvidf. taken up by this transporter in exchange for intracellular glutamate cea.fr and is then reduced to cysteine. Thus, this transport system is im- 3 Abbreviations used in this paper: EAAT, excitatory amino acid transporter; GSH, portant for maintaining intracellular glutathione (GSH) levels (22, glutathione; MDM, monocyte-derived macrophage; THA, DL-threo-␤-hydroxyaspartic acid; DHK, dihydrokainate; trans-PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; 23). An increase in extracellular glutamate concentration could Vmax, maximum velocity, Km, Michaelis constant; Ki, inhibition constant. thus deplete intracellular GSH by competing with cystine uptake

Ϫ do have an XAG glutamate transport system, which could change current thinking about the role of macrophages in the brain and raises the question as to the role of these transporters in the regulation of glutamate-driven immunoregulation in the periphery. Materials and Methods Human monocyte isolation and differentiation Human PBMC were isolated from the blood of healthy HIV-seronegative donors by Ficoll-Hypaque density gradient centrifugation. Monocytes were separated from PBMC by countercurrent centrifugal elutriation. Monocytes (2 ϫ 106 cells/well) were seeded in 48-well plates in RPMI 1640 medium (Boehringer Mannheim, Mannheim, Germany) supplemented with 10% heat-inactivated (56°C for 30 min) FCS (Boehringer Mannheim), 2 mM L-glutamine (Boehringer Mannheim), and 1% antibiotic mixture (penicil- lin, streptomycin, and neomycin; Life Technologies, Grand Island, NY).

Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. In our hands, blood monocytes (Ն95% enriched after elutriation) became adherent after1hofculture and then spontaneously detached from the plastic after 24 h and retained a monocyte-like appearance for 5 days (Fig. 1A). Monocytes were then washed with PBS and distributed in 48-well plates (0.5 ϫ 106 cells/well) in 10% FCS culture medium supplemented with 15% human PBMC-conditioned medium (7 days of culture). At day 7–8 of culture, cells tightly adhered to the plastic, and morphological differentiation occurred such that the monocyte-macrophages became fibroblast-like (Fig. 1B). At day 9–12, the cells became large, well-dispersed rounded macrophages, and they retained this appearance for about 25 days (Fig. 1C). Flow cytometric analysis of cell surface molecule expression Adherent cells were detached from the plastic by a 20-min incubation at 37°C in nonenzymatic cell dissociation solution (Sigma, Saint Quentin Fallavier, France). The cells were incubated for 30 min at 4°C with FITC- or PE-conjugated mAbs against CD14 (Becton Dickinson, Mountain View, CA), HLA-DR (Immunotech, Marseille, France), CD11b (Immunotech), CD16 (Immunotech), or irrelevant isotype-matched controls. The cells were washed twice with PBS, fixed in 200 ␮l PBS/1% paraformaldehyde (weight to volume ratio), and analyzed for fluorescence using a FACScan flow cytometer (Becton Dickinson). Viable cells were gated using a for- ward- and side-scatter pattern. HLA-DR and CD16 expression increased FIGURE 1. Morphological aspect of monocytes and macrophages dur- with cell differentiation (Table I). CD14 expression was up-regulated until ing in vitro differentiation. Elutriated monocytes were seeded in 48-well day 5, was undetectable in fibroblast-like macrophages (day 7), and was culture plates and allowed to differentiate. Culture medium was replaced highly expressed again in a small subpopulation of differentiated amoeboid twice weekly. A, Cells retained a monocytic appearance for 5 days. B, macrophages (day 12). Similarly, the CD11b adhesion receptor was absent Between days 7 and 8, tightly adherent cells exhibited fibroblastic mor- in day 5 monocytes but was strongly expressed in adherent macrophages. phology. C, Amoeboid morphology of large, rounded macrophages by days Obtention of tissular macrophages 9–12. Original magnification, ϫ64. Macrophages were purified from bronchoalveolar lavages performed in cynomolgus macaques after local anesthesia with lignocaide (Xylovet, (24, 25). Recent studies have demonstrated the presence of EAATs Sanofi, France), as previously described (31). Spleens were obtained from C57BL/6 mice. We also obtained spleen tissue from two splenectomized in nonneural cells (26–28), suggesting that extracellular glutamate patients, one after idiopatic thrombopenic purpura and one after clearance is required both centrally and outside the brain. Minkowski-Chauffard hemolytic anemia. Spleens were gently dissociated As tissue macrophages and microglia release glutamate upon in in isotonic NaCl with forceps and were sieved (100 ␮m). Mononucleated vitro stimulation (20, 21, 29, 30), we addressed the question of cells were isolated by Ficoll-Hypaque density gradient centrifugation. Sim- ϩ ian alveolar and murine splenic macrophages were purified by adhesion for whether these cells also possess Na -dependent high-affinity glu- 1 h to 48-well plates in RPMI 1640 with 10% FCS, 2 mM L-glutamine, and tamate transporters for clearing extracellular glutamate. Using hu- 1% antibiotic mixture (106 cells/wells). Human splenic cells were cultured man monocyte-derived macrophages, we showed that these cells for 5 days in RPMI 1640 with 10% FCS, 2 mM L-glutamine, and 1%

Table I. Cell surface molecule expression during differentiation of monocytes into macrophagesa

CD14 HLA-DR CD11b CD16

Day of Culture % Cells MFI % Cells MFI % Cells MFI % Cells MFI

0b 52 267 46 147 40 165 18 42 1b 70 289 79 510 88 177 80 372 5b 65 498 92 1640 0 0 59 181 7c 0 0 85 3326 71 630 42 131 12d 11 709 75 2296 57 593 54 523

a Percentage of positive cells and mean ﬂuorescence intensity (MFI) were obtained for each marker by subtracting isotype-matched negative control results. Results were rounded to the nearest integer. Data are from one representative experiment of four. b Cells with a monocytic morphology. c Cells with a ﬁbroblast-like morphology. d Cells with an amoeboid morphology. 5432 GLUTAMATE TRANSPORT IN MACROPHAGES

antibiotic mixture, and then nonadherent cells were removed. Glutamate uptake experiments were performed at days 9–12.

Microglial cell cultures Microglia were purified from mixed glial cultures from neonatal C57BL/6 mice, as previously described (32). Briefly, pieces of cortex from postnatal 1-day-old mice were incubated in trypsin and mechanically dissociated. Cells were plated and fed weekly with DMEM (Life Technologies), 4.5 g/L glucose, Glutamax-I, and 10% FCS. After 13–15 days of culture, microglial cells were dislodged from mixed glial cultures by shaking for2hat 220 rpm. Microglial cells were allowed to settle in 48-well plastic dishes for 30 min, after which the supernatant was replaced with DMEM, 10% FCS, and 2% B27 supplement (Life Technologies). Experiments were performed 6 days later, that is, 19–21 days after brain removal. Microglia- enriched cultures were more than 98% pure, as assessed by immunocyto- chemistry (Mac-1) and isolectin (B4) staining (data not shown). FIGURE 2. Glutamate uptake by MDM. MDM were incubated for 5 Primary mouse cortical neuron cultures min at 37°C or at 4°C in uptake medium containing various concentrations 3 Primary mouse cortical cells were cultured from 15-day C57BL/6 mouse of [ H]glutamate. Intracellular radioactivity was measured as described in embryos. Cortices were dissected under a binocular microscope, carefully Materials and Methods. A, Data are mean Ϯ SEM obtained with MDM freed of meninges, and incubated in trypsin/EDTA for 10 min at 37°C. from three donors. B, Data were plotted as a Lineweaver-Burk plot to Trypsin was inactivated by incubation in DMEM, 4.5 g/L glucose, Glu- obtain Vmax and Km for transport measured at 37°C. tamax-I, and 1% FCS. Cells were then dissociated mechanically in DMEM, 1% FCS, with a flame-narrowed Pasteur pipette. Cells were pelleted by centrifugation and resuspended in DMEM, 2% B27 and 3% FCS. Ninety- 4 once with PBS. Starvation and uptake were done as they were for macro- six-well plates coated with poly D-lysine were seeded at 7 ϫ 10 cells per phages. Uptake was stopped by washing twice with 5 ml of cold PBS. well in 150 ␮l of medium (DMEM/B27/FCS, with antibiotics). Cultures were kept at 37°C, 5% CO2 for 2 days. The medium was then replaced with Semiquantitative RT-PCR serum-free DMEM/B27/antibiotics. After 1 or 2 wk in culture, cells were immunocytochemically assessed to be Ͼ95% neurons (according to mi- mRNA levels were assessed by a noncompetitive RT-PCR method crotubule associated protein-2 immunolabeling) and less than 6% glial routinely used in our laboratory (33). Briefly, RNA was extracted using cells (5% glial fibrillary acidic protein-positive cells and less than 1% RNAble (Eurobio, les Ulis, France) according to the manufacturer’s in- MAC-1- or IB4-positive cells). struction. Total RNA was treated with 5 U RNase-free DNase (Boehringer Mannheim) for 45 min at room temperature, and DNase was then inacti- Neurotoxicity experiments vated by heating for 5 min at 95°C. RNA was reverse-transcribed in op- timal conditions, as previously defined (34, 35). Primers were as follows: Monocyte-derived macrophage (MDM; 8–12 days) were cultured in EAAT-1 sense, 5Ј-GCTAGATAGTAAGGCATCAGGGAA-3Ј; EAAT-1 DMEM containing 4.5 g/L glucose Glutamax-I without FCS at 37°C and antisense, 5Ј-AAGCACATGGAGAAGACAACTAGA-3Ј (amplicon size, ␮ ␮ 5% CO2 in the presence or absence of 100 M, 300 M, or 1 mM gluta- 429 bp); EAAT-2 sense, 5Ј-TGGATGCTAAGGCTAGTGGC-3Ј; EAAT-2 mate. Culture medium containing the same concentrations of glutamate antisense, 5Ј-GCACCTCAGTCACAGTCTCG-3Ј (amplicon size, 345 bp); was incubated in the same plate but in the absence of MDM as a control. EAAT-3 sense, 5Ј-TTCTAGGTATTGTGCTGGTGGTGA-3Ј; EAAT-3 Supernatants were harvested at 6 h, 20 h, or 4 days, centrifuged to eliminate antisense, 5Ј-TCCAAAGACAAGGCAAAAGACAAT-3Ј (amplicon Ϫ ␮ cell debris, and stored at 20°C. Aliquots (100 l) were tested in triplicate size, 350 bp); GAPDH sense, 5Ј-ACCACCATGGAGAAGGCTGG-3Ј; by incubation with 7-day-old primary mouse cortical cells for 24 h. Neuron GAPDH antisense, 5Ј-CTCAGTGTAGCCCAGGATGC-3Ј (amplicon viability was then measured by using the MTT assay (Sigma). Results were size, 509 bp). expressed in OD540–630. The percentage of neuroprotection afforded by Primer specificity was confirmed by both amplicon size assessment and ϭ ϫ MDM against glutamate was calculated as follows: % protection 100 restriction analysis with StuI, HindIII, and AVAII. RT-PCR amplicons were Ϫ [(MDM with glutamate) (medium with glutamate)/(MDM without glu- resolved in a 1.5% agarose gel by electrophoresis, and signal was quanti- Ϫ tamate) (medium with glutamate)]. fied with densitometric analysis software (NIH Image 1.2; W. Rasband, National Institutes of Health, Bethesda, MD). The relative abundance of Glutamate uptake mRNA species was determined using a standard curve for each PCR run. PCR was performed with three or four, one in four dilutions of each sam- Glutamate uptake was determined for MDM, simian alveolar macrophages, ple, giving a semilog range of amplification. Each amplification was re- murine microglia, and murine and human spleen macrophages, seeded in peated at least twice. Data are expressed as the ratio of the signal obtained 48-well plates. The uptake medium was 137 mM NaCl, 0.7 mM K HPO , 2 4 for each glutamate transporter divided by that obtained for GAPDH in the 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4). ϩ same sample, to permit the comparison of RNA species between samples. We assessed Na dependence by replacing the NaCl (137 mM) with 137 mM choline chloride (Sigma). Cells were washed with 1 ml PBS and Intracellular glutathione content incubated for 20 min at 37°C in 200 ␮l uptake medium with ionic modifications or inhibitors, if necessary, such as DL-threo-␤-hydroxyaspartic MDM were cultured overnight in DMEM without cystine, glutamine, and Ϫ Ϫ Ϫ acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC), dihy- glutamate (DMEM Cyst /Gln /Glu ; Life Technologies), supplemented drokainate (DHK), L-cystine, quisqualic acid, L-homocysteate, or L-␣-ami- with 0.1% FCS. Cells were then washed with PBS and incubated with 300 noadipate (Sigma). The medium was aspirated and replaced with 100 ␮l ␮l DMEM CystϪ/GlnϪ/GluϪ supplemented with 0.1% FCS in the presence uptake medium (with ionic modifications or inhibitors, if necessary) con- or absence of cystine, glutamate, or THA for 4.5 h. MDM were washed 3 taining L-[2,3- H]glutamic acid (30–60 Ci/mmol; ICN, Irvine, CA). For with PBS and lysed with 150 ␮l PBS, 0.1% Tween for 1 h. GSH content concentrations above 50 ␮M, [3H]glutamate specific activity was reduced was measured using an enzymatic assay (Cayman Chemicals, Ann Arbor, by a factor of 100 or 200 by adding unlabeled glutamate (Sigma). Uptake MI) as specified by the manufacturer. Protein content of cell lysates was was stopped after 5 min by removing medium and washing twice with 1 ml determined by the Bradford method. PBS. Cells were then lysed with 130 ␮l of 100 mM NaOH. The radioactivity of 60 ␮l of lysate was determined by liquid scintillation counting. Results The protein content of 60 ␮l of cell lysate was determined by the Bradford method. All experiments were performed in triplicate. Glutamate uptake is Glutamate uptake by MDM expressed as picomoles of glutamate per milligram of protein per minute. MDM were incubated with various concentrations of [3H]gluta- Glutamate uptake into nonadherent monocytes was also assessed: monocytes were cultured in RPMI 1640, 10% FCS, with or without various mate for 5 min at 4°C or at 37°C, and cell-associated radioactivity doses of TNF-␣ (R&D Systems, Minneapolis, MN). Cells were harvested, was measured (Fig. 2A). Cell-associated radioactivity was about dispensed into 12-ml polypropylene tubes (106 cells/tube), and washed 90% lower for incubations at 4°C than for those at 37°C, consistent The Journal of Immunology 5433

Table II. Inhibition of glutamate uptake by EAA analoguesa

␮ Compound % Inhibition Ki ( M) THA 94.2 Ϯ 0.3 15 Ϯ 5 trans-PDC 88.7 Ϯ 0.3 56 Ϯ 33 Dihydrokainate 25.6 Ϯ 2.53 Ͼ1000 L-␣-Aminoadipate 29.5 Ϯ 10 Ͼ1000 Quisqualate 19.9 Ϯ 5 Ͼ1000 L-Cystine 16.6 Ϯ 11 Ͼ1000 L-Homocysteate 13.7 Ϯ 5.7 Ͼ1000

a MDM were incubated in uptake buffer containing 1 ␮M glutamate and the tested inhibitor for 5 min at 37°C. Intracellular radioactivity was measured as described in Materials and Methods. Values represent percent inhibition by 1 mM inhibitor. Ki was determined using glutamate concentrations ranging from 1 to 1000 ␮M. Data are mean Ϯ SEM obtained with MDM from three donors.

Inhibition of MDM glutamate uptake by EAA analogues We assessed the potential of EAA analogues for inhibiting glutamate uptake by MDM (Table II). THA and trans-PDC, two com- petitive inhibitors specific for EAATs, efficiently inhibited 1 ␮M Ϯ glutamate uptake with inhibition constant (Ki) values of 15 5 and 56 Ϯ 33 ␮M, respectively. DHK and L-␣-aminoadipate, which inhibit EAAT2 but not EAAT1, inhibited uptake by only 25.6 Ϯ 2.5% and 29.5 Ϯ 10%, respectively, when present in a 1000-fold excess over glutamate. L-cystine, L-homocysteate, and quisqualate FIGURE 3. Ionic dependence of glutamate uptake by MDM. A, MDM reduced glutamate transport by 16.6 Ϯ 11%, 13.7 Ϯ 5.7%, and were incubated for 5 min at 37°C with various concentrations of [3H]glu- 19.9 Ϯ 5%, respectively. Thus, glutamate uptake by MDM is tamate in uptake medium containing 137 mM NaCl or 137 mM choline mostly mediated by a THA- and trans-PDC-sensitive transport sys- chloride. Data are one typical experiment of three. Data are expressed as tem, such as EAAT1 or EAAT3, but probably not by EAAT2. At Ϯ mean SD of triplicate results. B, MDM were incubated for 5 min with low glutamate concentrations, only 15% of glutamate uptake was 1 ␮M[3H]glutamate in uptake medium, with or without CaCl or various 2 abolished by blocking the cystine/glutamate transporter, demon- concentrations of NaCl, choline chloride, or KCl (mM). Data are mean Ϯ strating the higher affinity of glutamate for EAATs over the cys- SEM obtained with MDM from three donors. tine/glutamate antiporter.

Time-course of Naϩ-dependent high-afﬁnity glutamate with glutamate transport by MDM rather than receptor binding. At transporter expression and function during culture of 37°C, intracellular radioactivity linearly increased with time for at monocyte-macrophages least 5 min and was within 10 and 20% of linearity after 10 min (data not shown). The values obtained at 5 min were thus consid- EAAT1 and EAAT2 genes were weakly expressed or undetectable ered to be satisfactory approximations of initial uptake rates. We on freshly sorted monocytes (Figs. 4 and 5A). EAAT1 and EAAT2 determined the kinetic parameters of glutamate uptake by MDM mRNA expression levels markedly increased after1hinculture, by measuring initial uptake velocities at 37°C for glutamate con- reached a maximum by day 2 (120,000- and 55,000-fold increases, centrations of 1–400 ␮M. Glutamate uptake was found to be sat- respectively), and then slowly decreased until day 12 (Fig. 5A). We urable and approached saturation at 150 ␮M. Lineweaver-Burk found no change in EAAT3 mRNA levels over time (weak signal, Ϯ ␮ plots (Fig. 2B) revealed a Michaelis constant (Km)of77 6 M Ϯ and a maximum velocity (Vmax) of 2044 181 pmol/mg protein/min.

Ionic requirements of glutamate uptake by MDM We determined the glutamate transport in uptake medium with ionic modifications. We first showed that the absence of sodium inhibited glutamate uptake by 80 and 70% for glutamate concentrations of 1 and 200 ␮M, respectively (Fig. 3A). The mean Ϯ SEM sodium dependence for nine donors was 68 Ϯ 4.7%, with a range of 50–88% (see Fig. 7). The effects on glutamate uptake of changing Kϩ and Ca2ϩ concentration were also evaluated. In- creasing Kϩ concentration from 1.4 mM to 68.5 mM reduced uptake by 60 Ϯ 5% compared with the value obtained when Kϩ was replaced by an equimolar concentration (68.5 mM) of choline FIGURE 4. EAAT1 and EAAT2 mRNA levels in blood monocytes and chloride (Fig. 3B). These observations suggest that 60–80% glu- MDM. Monocyte mRNA was extracted just after elutriation. MDM mRNA tamate uptake by MDM is mediated by the Naϩ/Kϩ-dependent Ϫ 2ϩ was extracted after 10 days of differentiation. Amplification was performed high-affinity glutamate transport (XAG ). The absence of Ca with 4- or 5-fold dilutions of cDNA. Numbers of PCR cycles were 38 for had no effect on glutamate uptake, suggesting that this transport EAAT1 and EAAT2 and 31 for GAPDH. Positive controls were amplicons 2ϩ mechanism is different from the Ca -dependent glutamate uptake from primary cultures of simian astrocytes. The size of each band is indi- also described for brain homogenates (36). cated in base pairs. 5434 GLUTAMATE TRANSPORT IN MACROPHAGES

FIGURE 6. Effect of TNF-␣ on glutamate transport by monocytes. Freshly elutriated monocytes were cultured in RPMI 1640, 10% FCS, with or without various concentrations of TNF-␣ for 3 days. The 100-␮M [3H]glutamate uptake assays were performed as described in Materials and Methods in uptake medium with Naϩ (137 mM NaCl) or without Naϩ (137 mM choline chloride) at 37°C. The values given are for Naϩ-dependent glutamate transport, calculated by subtracting Naϩ-independent uptake (w/o Naϩ) from total uptake (with Naϩ). Data are from one of three experiments. Data are expressed as means Ϯ SD of triplicate determinations.

(152 Ϯ 9 vs 1123 Ϯ 4 pmol/mg protein/min for 0 and 100 ng/ml TNF-␣, respectively) and the appearance of the Naϩ-dependent glutamate transport (859 pmol/mg protein/min for monocytes stimulated with 100 ng/ml TNF-␣) (Fig. 6). This suggests that FIGURE 5. Time course of EAAT1 and EAAT2 mRNA expression and circulating monocytes may rapidly acquire functional EAATs in glutamate uptake during in vitro culture of monocyte-macrophages. A, Hu- inﬂammatory conditions. man monocyte-macrophages were cultured for 12 days and mRNA was extracted at various times for RT-PCR. Results are expressed in arbitrary Glutamate uptake by in vivo-differentiated macrophages and units as described in Materials and Methods. Data are from one of two microglia experiments, expressed as mean Ϯ SD of triplicate determinations. B, Glu- tamate uptake assays were performed with monocytes (1 ϫ 106 cells/tube, We investigated whether tissue macrophages and microglia trans- nonadherent cells) or MDM (adherent cells). Cells were incubated with 100 port glutamate (Fig. 7). No signiﬁcant glutamate transport was ϩ ␮M[3H]glutamate for 5 min in uptake medium with Na (137 NaCl) or detected for murine splenic and simian alveolar macrophages the ϩ Ϯ without Na (137 mM choline chloride) at 37°C. Data are mean SEM day of isolation (about 250 and 160 pmol/mg protein/min for total obtained with MDM from two donors.

data not shown). On the day of elutriation, the monocytes did not transport significant amounts of glutamate (71 Ϯ 3 pmol/mg protein/min) (Fig. 5B). An increase in Naϩ-independent glutamate uptake was observed after 20 h of culture (580 Ϯ 122 and 536 Ϯ 196 pmol/mg protein/min for total and Naϩ-independent glutamate uptake, respectively). Naϩ-independent glutamate transport then decreased slowly with time, reaching 237 Ϯ 10 pmol/mg protein/min after 5 days. Naϩ-dependent glutamate transport (1,347 Ϯ 107 pmol/mg protein/min) and an increase in Naϩ-independent transport (1,271 Ϯ 295,107 pmol/mg protein/min) be- gan on day 8, concomitant with the morphological differentiation of monocytes into fibroblast-like macrophages. Total glutamate uptake then slowly decreased between days 8 and 14, and there were no further changes in the two systems of glutamate transport from day 14 to day 30 (data not shown). FIGURE 7. Glutamate uptake by tissue macrophages, microglia, and MDM. Murine spleen macrophages and simian alveolar macrophages were Effect of TNF-␣ on glutamate transport by monocytes seeded in 48-well plates, and a glutamate uptake assay was performed after ␣ 1 h of adherence. Human MDM and human spleen macrophage uptake We investigated whether TNF- , an inflammatory cytokine assays were performed after 9–12 days of culture. Murine microglia uptake present at high concentration in both the periphery and CNS in assays were performed after 20 days of culture. Macrophages were incu- many diseases, could induce glutamate transport by monocytes. bated with 100 ␮M[3H]glutamate for 5 min in uptake medium containing Three-day stimulation of freshly elutriated monocytes induced a 137 mM NaCl or 137 mM choline chloride at 37°C or in uptake medium ϩ dose-dependent increase in Na -independent glutamate transport at 4°C. Data are mean Ϯ SEM. n, Number of independent experiments. The Journal of Immunology 5435

uptake and glutamate binding, respectively). Nevertheless, we detected efﬁcient glutamate transport in 9- to 12-day cultured human splenic macrophages and in 20-day cultured murine microglia that was 79 Ϯ 3.2% and 76 Ϯ 7% Naϩ-dependent, respectively, and had a velocity similar to that of human MDM (1469 Ϯ 92 and 2230 Ϯ 433 pmol/mg protein/min for 100 ␮M glutamate, respectively). This shows that tissue macrophages originating from both the CNS and peripheral organ do express functional EAATs after some days in culture.

Effect of MDM on glutamate-induced neurotoxicity We tested whether MDM could regulate extracellular glutamate concentration by culturing MDM for 6 h, 20 h, or 4 days in the presence of various concentrations of glutamate. Supernatants were harvested and tested for neurotoxicity using primary mouse neuronal cultures. We also assessed the neurotoxicity of these glutamate concentrations in culture medium without MDM as a control (Fig. 8). As previously described, we observed that supernatants from macrophages without glutamate induced a neurotoxicity, which was maximal after6hofculture (36%) and decreased to 26% and 18% after 20 h and 4 days of culture, respectively. In our culture conditions, 100 ␮M glutamate was sufficient for maximal toxicity to neurons (50%). No significant degradation of glutamate was observed in culture medium at 37°C (in the absence of MDM), as neurotoxicity was similar for all incubation times. MDM induced a time- and dose-dependent protection against glutamate neurotoxicity. After6hofculture (Fig. 8A), MDM reduced the neurotoxicity induced by 100 ␮M glutamate by 43%. After 20 h of culture (Fig. 8B), MDM reduced glutamate-induced neurotoxicity by 62.5, 77.5, and 36% for 100, 300, and 1000 ␮M glutamate, respectively. After 4 days of culture (Fig. 8C), maximum protection against glutamate neurotoxicity was obtained: 100, 81, and ␮ 41% for 100, 300, and 1000 M glutamate, respectively. Vmax for Naϩ-dependent glutamate uptake by MDM in this experiment was 3000 pmol/mg protein/min (uptake measured for 5 min, data not shown). This velocity would result in the clearance of 100% of 100 and 300 ␮M glutamate and 32% of 1 mM glutamate after 4 days of culture. These values are consistent with the observed kinetics FIGURE 8. Effect of MDM on glutamate-induced neurotoxicity. MDM of the neuroprotective effect of MDM. Fig. 8D shows the mean Ϯ were incubated in DMEM without FCS, with or without glutamate (100 SEM percentage of neuroprotection induced by MDM against glu- ␮M, 300 ␮M, or 1 mM) at 37°C. Culture medium containing these glutamate toxicity in two independent experiments. The results are tamate concentrations was incubated on the same plate as controls. Super- similar to those shown in Fig. 8, A–C. natants were harvested after6h(A), 20 h (B), or 4 days (C) and tested for neurotoxicity as described in Materials and Methods. Data are from one of two experiments and data are expressed as means Ϯ SD of triplicate results. D, Mean percentage neuroprotection afforded by MDM against the Effect of extracellular glutamate and glutamate transport on various concentrations of glutamate. Data are means Ϯ SEM of two inde- intracellular GSH concentration pendent experiments. Because cystine and glutamate are precursors for GSH synthesis, we tested the ability of these two amino acids to modulate GSH synthesis. A weak level of intracellular GSH (about 40 nmol/mg protein) was measured in MDM cultured in the absence of cystine. Discussion Cystine (100 ␮M) induced GSH synthesis (73 Ϯ 2 nmol/mg pro- We demonstrated in this study that macrophages derived from hu- tein), and this level was increased by 43% with 100 ␮M exogenous man blood monocytes have both a Naϩ-independent and a Naϩ- glutamate (104 Ϯ 3 nmol/mg protein). The level of intracellular dependent (67.7 Ϯ 4.7%) glutamate transport system. This latter GSH in cystine- and glutamate-incubated cells returned to a value was inhibited by an increase in extracellular Kϩ concentration. of 35 nmol/mg protein when MDM were incubated with 5 mM of This is consistent with the mechanism proposed by Kanner (37) for ϩ ϩ L-buthionine-[S,R]-sulfoximine, a blocker of ␥-glutamyl-cysteine- the Na /K high-affinity uptake of glutamate in the CNS. Gluta- synthetase (data not shown). When MDM were cultured in the mate uptake would indeed be driven by a Naϩ concentration gra- presence of cystine, glutamate, and THA (1 mM), intracellular dient and by a Kϩ concentration gradient in the opposite direction. GSH level returned to a value of 58 Ϯ 6 nmol/mg protein. This However, we cannot exclude that increased extracellular Kϩ may demonstrates that EAATs are indeed involved in the regulation of also act through disturbance of the Naϩ gradient as it depolarizes intracellular GSH synthesis by MDMs by providing intracellular the membrane potential and reduces the electrical component of glutamate. the electrochemical gradient for Naϩ entry. Because uptake was 5436 GLUTAMATE TRANSPORT IN MACROPHAGES insensitive to Ca2ϩ, it clearly differs from the Ca2ϩ/ClϪ-dependent glutamate uptake previously described in brain homogenates (36). At low glutamate concentration (1 ␮M), glutamate uptake was dose-dependently inhibited by the EAAT inhibitors THA and Trans-PDC, whereas a 1000-fold molar excess of L-cystine (or L-homocysteate) over glutamate inhibited glutamate uptake by only 15–20%. This demonstrates that MDM express EAATs and Ϫ that extracellular glutamate is mostly taken up by the XAG sys- Ϫ tem rather than the Xc system. In this study, numerous lines of evidence suggest that EAAT1 would be largely responsible for Naϩ-dependent glutamate transport by MDM: 1) DHK, an EAAT2-specific inhibitor, and L-␣- aminoadipate, an EAAT2 and EAAT4-specific inhibitor weakly inhibited glutamate uptake (Յ30%) (9, 38, 39); and 2) EAAT3 mRNA was barely detectable in MDM. However, we cannot rule out the involvement of the recently cloned EAAT5 because spe- FIGURE 9. Effect of glutamate and EAAT inhibitor on GSH content of MDM. MDM were cultured in DMEM CystϪ/GlnϪ/GluϪ supplemented cific inhibitors of this transporter have not been yet identified. The with 0.1% FCS in the presence or absence of 100 ␮M cystine, 100 ␮M Ϯ ␮ Ϯ Km and Vmax for glutamate transport were 77 6 M and 2044 glutamate, or 1 mM THA for 4.5 h. MDM were lysed with 150 ␮l PBS, 181 pmol/mg protein/min, respectively, for total uptake and 0.1% Tween 20 for 1 h, and GSH content was measured using an enzy- ϩ 58.5 Ϯ 23 ␮M and 1333 Ϯ 471 pmol/mg protein/min for Na - matic assay. Data are from one of three experiments. Data are expressed as dependent uptake. These values are very similar to those obtained means Ϯ SD of triplicate determinations. by others for cortical neurons, cortical synaptosomes, and glial cultures (40–42). Therefore, MDM may be as efficient as neural cells (astrocytes and neurons) in clearing extracellular glutamate, It is known from antisense-based knockout studies that EAAT1 suggesting an important role in the CNS. and EAAT2 are critical for the regulation of extracellular glutamate concentrations in the brain (14, 15). The extracellular con- Time-course studies demonstrated a large increase in EAAT1 centration of glutamate significantly increases in many neurolog- and EAAT2 mRNA levels at an early stage of monocyte culture. ϩ ical disorders (for review, see Ref. 2). This may be due to an Cultured monocytes did not display Na -dependent glutamate ϩ increase in intrathecal glutamate production (including by acti- transport until day 5, but we detected a Na -independent gluta- vated macrophages and microglia) and/or to the down regulation mate transport after 20 h of culture that was probably mediated by ϩ of astroglial glutamate transporters, especially through macroph- the cystine/glutamate transporter. Na -dependent transport coin- age-produced mediators such as arachidonic acid, oxygen-free rad- cided with the morphological differentiation of monocytes into fi- icals, or TNF (19, 20, 46–49). We indeed observed that MDM broblast-like cells, and a high level of transport was maintained constitutively produce neurotoxins, but we also showed that MDM until day 30 of culture. Such a dissociation of EAAT gene expres- time- and dose-dependently clear glutamate from culture medium, sion and glutamate transporter activity has already been described thereby reducing excitotoxicity. Our results thus suggest that dur- (for review, see Ref. 43). It would be of interest to investigate the ing brain injury associated with an increase in extracellular gluta- possible posttranscriptional regulation or regulation of signal mate concentration, infiltrating and resident macrophages, al- transduction mechanisms, accounting for differences between gene though producing neurotoxins, may also, together with astrocytes, expression and transporter activity in MDM. Indeed, Casado et al. regulate extracellular glutamate levels. These observations are in (44) showed that protein kinase C-dependent phosphorylation in- line with a recent study demonstrating that EAAT activity, al- duced an increase of EAAT activity, and Dowd and Robinson (45) though detectable in embryonic and early postnatal astrocytes, de- described the PMA induction of EAATs in cycloheximide-treated cline to undetectable levels in mature astrocytes (16). This sug- C6 cells, which was therefore independent of protein synthesis. gests that the relative roles of astrocytes and macrophages/ We did not detect any glutamate transport by in vivo-differen- microglia in the regulation of extracellular glutamate tiated macrophages from spleen or lung on the day of cell isolation. concentrations might be reconsidered and should be more pre- This is consistent with previous reports (29, 30) suggesting that cisely studied with a developmental perspective. resting tissue macrophages do not constitutively express functional Cystine, cysteine, and glutamate are GSH precursors, and EAATs. Nevertheless, our in vitro data show that MDM activated Reichelt et al. (50) reported in retinal Muller glial cells that GSH by adhesion to plastic before differentiation, as well as cultured synthesis could be limited by the capacity of EAATs to provide human splenic macrophages and cultured microglia, do have a intracellular glutamate for both cystine uptake and direct insertion Ϫ highly efficient XAG transport system. This provides evidence into GSH. The presence of both the cystine/glutamate transporter that, in the CNS, both resident (microglia) and infiltrating macro- and EAATs on MDM, the higher affinity of glutamate for EAATs, ϩ phages acquire high-affinity Na -dependent glutamate transporters and our data (Fig. 9) demonstrating that uptake of glutamate via Ϫ and lower excitotoxicity. Alternatively, XAG transport may also EAATs indeed increases GSH synthesis induced by cystine sug- be induced in the periphery by specific stimulation by cytokines or gest that there is a continuous glutamate exchange between intra inflammatory mediators. This second possibility is supported by and extracellular media. Glutamate is probably excreted through ϩ our results demonstrating that TNF-␣ stimulation induces Na - the cystine/glutamate transporter in exchange for cystine, leading dependent glutamate transport by monocytes and that 9- to 12-day to a decrease in its intracellular concentration. In turn, EAATs may Ϫ cultured splenic macrophages do have efficient XAG transport take up extracellular glutamate, thus limiting the extracellular systems. Additional studies of the stimulation requirements for competition between glutamate and cystine for the cystine/gluta- Ϫ XAG system expression or regulation in monocytes and macro- mate antiporter and maintaining intracellular glutamate availabil- phages and in situ expression of EAATs are required to support ity. 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