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 excito- toxicity 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 Arme´es, 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-␤-hydroxyaspar- tic 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 Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 5431 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 reg- ulation 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 adher- ent 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 differenti- ation occurred such that the monocyte-macrophages became fibroblast-like (Fig. 1B). At day 9–12, the cells became large, well-dispersed rounded mac- rophages, 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.