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C57/BL6 mice (Charles River laboratories, Wilmington, MA). Cells were isolated ...... Roussel, M. F., Cleveland, J. L., Shurtleff, S. A., and Sherr, C. J.. (1991) Myc ...
Macrophages require different nucleoside transport systems for proliferation and activation ´ SOLER,* JOSE´ GARCI´A-MANTEIGA,‡ RAQUEL VALDE´S,‡ JORDI XAUS,* CONCEPCIO ` MONICA COMALADA,* F. JAVIER CASADO,‡ MARC¸AL PASTOR-ANGLADA,‡,1 ANTONIO CELADA,*,1 AND ANTONIO FELIPE‡,1,2 *Departament de Fisiologia (Biologia del Macro`fag) and Fundacio´ August Pi i Sunyer, Campus de Bellvitge; and ‡Departament de Bioquı´mica i Biologia Molecular, Universitat de Barcelona, E-08028 Barcelona, Spain To evaluate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow-derived macrophages, the nucleosides required for DNA and RNA synthesis are recruited from the extracellular medium. M-CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon ␥ (IFN-␥) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M-CSF only up-regulated the equilibrative system es. Inhibition of this transport system blocked M-CSF-dependent proliferation. Treatment with IFN-␥ only induced the concentrative N1 and N2 systems. IFN-␥ also down-regulated the increased expression of the es equilibrative system induced by M-CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleoside transporters are critical for macrophage proliferation and activation.—Soler, C., Garcı´a-Manteiga, J., Valde´s, R., Xaus, J., Comalada, M., Casado, F. J., Pastor-Anglada, M., Celada, A., Felipe, A. Macrophages require different nucleoside transport systems for proliferation and activation. FASEB J. 15, 1979 –1988 (2001) ABSTRACT

Key Words: nucleoside uptake 䡠 interferon ␥ 䡠 macrophage colony-stimulating factor

The mononuclear phagocyte family comprises numerous cell types, including tissue macrophages, liver Kupffer cells, dermal Langerhans cells, bone osteoclasts, brain microglia, and perhaps some of the interdigitating and follicular dendritic cells from lymphoid organs (1). Macrophages originate from undifferentiated stem cells in the bone marrow and through the blood reach the different tissues where, in most cases, they undergo apoptosis. In the presence of specific growth factors or cytokines, macrophages proliferate, become activated, or differentiate. In response to the macrophage-colony stimulating factor (M-CSF), 0892-6638/01/0015-1979 © FASEB

macrophages are able to proliferate in the tissues (2, 3). To carry out their functional activities, macrophages must become activated. After interacting with interferon ␥ (IFN-␥), a cytokine released by activated T lymphocytes, macrophages undergo biochemical and morphological modifications that allow them to perform their functional activity (4). IFN-␥ blocks their proliferation and protects them from apoptosis (5, 6). Cell proliferation and activation involve high nucleic acid synthesis rates. Nucleosides from the extracellular milieu are the main source for nucleic acid synthesis and cross the plasma membrane through several transport systems. Depending on the cell type, different equilibrative and concentrative transport activities may be coexpressed. The equilibrative uptake is mediated by two transport proteins, ENT1 and ENT2, which encode for the es and ei transport activities, respectively, as revealed by their sensitivity to nitrobenzylthioinosine (NBTI), a nucleoside analog that specifically inhibits es activity at the nanomolar range (7–10). In addition, there is evidence for five concentrative activities (N1 to N5). N1, N2, and N3 correspond to transport agencies involved in purine, pyrimidine, and broad specificity transport activities, respectively (7–10). N4 has been reported only in the human kidney (11). N5 activity, which shows a purine-specific, guanosine-preferring uptake, has been described in mammalian cells of lymphoid origin and is sensitive to NBTI (12–14). Three gene products have been identified: CNT1, which corresponds to N2 (pyrimidine-preferring); CNT2, also named SPNT, which encodes for N1 (purine-preferring) (7–10); and very recently CNT3, which putatively encodes for N3 (broad specificity) (15). Hematopoietic cells are deficient in endogenous nucleotide synthesis and so require uptake of exogenous nucleosides to meet their metabolic demands (7, 8). Therefore, nucleoside uptake may be a key regulatory step for physiological responses in macrophages. We wanted to study how M-CSF-induced proliferation 1

M.P.A., A.C., and A.F. contributed equally to this study. Correspondence: Laboratori de Fisiologia Molecular, Departament de Bioquı´mica i Biologia Molecular, Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain. E-mail: [email protected] 2

1979

and IFN-␥-induced activation affect nucleoside transport activity. To this end, we used bone marrow-derived macrophages, which are nontransformed cells that respond to both proliferative and activating stimuli separately. We show that macrophages express both concentrative and equilibrative nucleoside transport systems. Furthermore, M-CSF and IFN-␥ regulate nucleoside transporters differentially according to specific requirements for DNA or RNA. Whereas M-CSF specifically up-regulates the expression of the equilibrative es system, IFN-␥ increases the concentrative N1 and N2 systems and down-regulates the equilibrative es system induced by M-CSF. The inhibition of the system es blocks M-CSF-dependent proliferation, which shows that this nucleoside transport system is essential to macrophage proliferation.

MATERIALS AND METHODS cDNA probes, antibodies, and reagents cDNA coding for the isoforms of nucleoside transport systems was generated by RT-PCR from rat tissues, as described elsewhere (16, 17). The cDNA fragments for CNT1 (bp 717 to 1280), CNT2 (bp 776 to 1465), ENT1 (bp 1 to 1375), and ENT2 (bp 356 to 1379) were blunted and subcloned in an EcoRV-digested Bluescript KS. The pBR18S plasmid was supplied by Dr. I. Fabregat (Universidad Complutense de Madrid, Spain). The specific anti-CNT1 antibody was generated and characterized against rat CNT1 peptide as reported earlier (16). This antibody cross-reacted against mouse CNT1 protein as described below and the specificity was validated by using a preimmune serum from the same rabbit (not shown). The ␤-actin antibody was purchased (Santa Cruz Biotechnology, Santa Cruz, CA). [3H]Uridine, [3H]guanosine, [3H]cytidine, [3H]-thymidine, and [␣-32P]dCTP were from Amersham Pharmacia Biotech (Amersham, Buckinghamshire, UK). Nonradioactive nucleosides and analogs were from Sigma (St. Louis, MO). DMEM and other culture medium reagents were from BioWhittaker (Verviers, Belgium). Klenow fragment and other molecular biology reagents were from Amersham Pharmacia. IFN-␥ was kindly provided by Genentech, Inc. (South San Francisco, CA). All other chemical reagents were of the highest quality from Sigma, Merck (Darmstadt, Germany) or Fluka Chemie (Buchs, Switzerland). Animals and cell culture The identification, characterization, and regulation of nucleoside transport system studies were performed in bone marrow-derived macrophages from 6- to 10-wk-old BALB/c or C57/BL6 mice (Charles River laboratories, Wilmington, MA). Cells were isolated and cultured as described elsewhere (18). Animals were killed by cervical dislocation and both femurs were dissected removing adherent tissue. The ends of the bones were cut off and the marrow tissue was flushed by irrigation with media. The marrow plugs were passed through a 25 gauge needle for dispersion. The cells were cultured in plastic dishes (150 mm) in DMEM containing 20% FBS and 30% L cell conditioned media as a source of M-CSF. Macrophages were obtained as a homogeneous population of adherent cells after 7 days of culture and maintained at 37°C in a humidified 5% CO2 atmosphere. They were cultured 1980

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with the same tissue culture differentiation media (DMEM, 20% FBS, 30% L cell medium) or arrested at G0 by M-CSF deprivation in a DMEM medium supplemented with 10% FBS for at least 18 h (starvation). When indicated, G0-arrested cells were incubated in the absence or the presence of M-CSF (1200 U/ml), with or without IFN-␥ (300 U/ml). All animal handling was approved by the ethics committee from the University of Barcelona. Transport experiments Macrophages were scraped 7 days after isolation and homogeneously seeded in 35 mm tissue culture dishes. Uptake was measured as described elsewhere with some modifications (17, 19). Transport was initiated by adding 1 ␮M of 3Hnucleoside (1 ␮Ci/ml) in either 137 mmol/l NaCl or 137 mmol/l choline chloride medium also containing 5.4 mmol/l KCl, 1.8mmol/l CaCl2, 1.2 mmol/l MgSO4, and 10 mmol/l HEPES (pH 7.4). At established times (usually 3 min), monolayers were washed three times in 2 ml of ice-cold buffer consisting of 137 mmol/l NaCl and 10 mM HEPES, pH 7.4. Cells were dissolved in 0.5 ml of 0.5% Triton X-100; 0.4 ml of cell extract was counted for radioactivity and the rest was used for protein determination according to Bradford (Bio-Rad laboratories, Madrid, Spain). Concentrative (Na⫹-dependent) uptake was calculated as the transport rate in the presence of Na⫹ minus the rate in its absence (choline medium). DNA and RNA synthesis and cell number DNA synthesis was measured as the incorporation of 3H-thymidine into DNA as described earlier (5) with minor modifications. To analyze the effect of IFN-␥ and M-CSF on DNA synthesis, macrophages (5⫻104) were seeded in 24-well plates in 1 ml of medium without M-CSF for at least 18 h (starvation). Cells were then cultured in the absence or presence of M-CSF with/without IFN-␥. At established times, the medium was removed and replaced by 0.5 ml of the same medium containing 1 ␮Ci/ml [3H]-thymidine; after three additional hours of incubation, cells were fixed in 70% methanol. They were washed three times in ice-cold 10% trichloroacetic acid and solubilized in 1% SDS and 0.3% NaOH. The entire contents of the well were used for counting radioactivity. In parallel experiments, RNA synthesis was measured as the incorporation of 3H-uridine into RNA (20) with minor modifications, as described above for DNA synthesis. The number of viable cells for each condition was measured by trypan blue exclusion with a hemocytometer. Total RNA isolation and Northern blot analysis Total RNA from mouse macrophages and rat hepatocytes was isolated using the RNeasy Mini kit from Qiagen (Hilden, Germany), following the manufacturer’s instructions. Up to 20 ␮g of total RNA was size-fractionated through 1% agarose, 3% formaldehyde gels in 20 mM 3-[N-morpholino]-propane sulfonic acid and 1 mM EDTA, pH 7.4. Equality of RNA loading in each lane was confirmed by addition of ethidium bromide to the samples before electrophoresis. RNA was transferred to an Immobilon filter (Amersham Pharmacia Biotech) by capillarity in 20⫻SSC (3M NaCl, 300 mM sodium citrate, pH 7.0). RNA was cross-linked to the filter by irradiation with UV light. Filters were treated, prehybridized, and hybridized at high stringency as described (21). The washing conditions for the cDNA probes (CNT1, CNT2, ENT1, ENT2, and 18S) were as described in refs 16, 17. Hybridization with the mouse 18S ribosomal RNA was used as a loading and transfer control.

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Protein extraction and Western blot analysis Cells were washed twice in cold PBS and lysed on ice with lysis solution (1% Nonidet P-40, 10% glycerol, 50 mmol/l HEPES pH 7.5, 150 mmol/l NaCl) supplemented with 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 86 ␮g/ml iodoacetamide, and 1 mM PMSF as protease inhibitors. The protein concentration of the samples was determined by Bio-Rad protein assay according to Bradford (22). The proteins from cell lysates (100 ␮g) were boiled at 95°C in Laemmli SDS loading buffer and separated on 10% SDS-PAGE. They were then transferred to nitrocellulose membranes (Hybond-ECL, Amersham Pharmacia Biotech). Protein expression analysis and generation and use of the CNT1 antibody were performed as described previously (16).

from macrophages was used in the RT-PCR reactions. The PCR. annealing temperature was set to 55°C during 10 cycles. The m18S rRNA oligonucleotides derived from the published sequence (24) were F (5⬘-CGCAGAATTCCCACTCCCGACCC3⬘, bp 482 to 498) and R (5⬘-CCCAAGATCCAACTACGAGC-3⬘, bp 694 to 675). Ten ␮l from the final RT-PCR reactions were electrophoresed in a 1% agarose gel (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0).

RESULTS Regulation of DNA and RNA synthesis by M-CSF and IFN-␥

Analysis of DNA content with DAPI Up to 106 macrophages from M-CSF-starved, treated with M-CSF (1200 U/ml), or M-CSF plus 100 nM NBTI were resuspended and fixed in ice-cold 70% ethanol. Cells were washed in PBS, solubilized in 0.2 ml of a solution containing 150 mM NaCl, 80 mM HCl, and 0.1% Triton, and incubated at 0 – 4°C for 10 min. Thereafter, 1 ml of a solution containing 180 mM Na2HPO4, 90 mM citric acid, and 2 ␮g/ml DAPI (pH 7.4) was added to each sample (5). After incubating the cells at 4°C for 24 h, their fluorescence was measured with an Epics Elite flow cytometer (Coulter, Fullerton, CA). A UV laser with an excitation beam of 25 mW at 333–364 nm was used and fluorescence was collected with a 525 nm band-pass filter. Cell doublets were gated out by comparing the area and width of the pulse. About 12,000 cells were counted for each histogram and cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems). RT-PCR analysis CNT1 mRNA expression was obtained by nested PCR as described earlier (16) using CNT1 oligonucleotides derived from rat intestine cDNA (23). Briefly, 1 ␮g of murine macrophage total RNA was incubated for 30 min at 42°C in one tube containing Ready to Go RT-PCR beads (Amersham Pharmacia Biotech). Individual 50 ␮l of RT-PCR reactions contained two units of Taq DNA polymerase, 10 mM Tris-HCl, (pH 9.0), 60 mM KCl, 1.5 mM MgCl2, 200 ␮M of each dNTP, Moloney murine leukemia virus reverse transcriptase, RNAguard ribonuclease inhibitor (porcine), and stabilizers including RNAse/DNAse-free BSA. Selected first reaction CNT1 primers at 1 ␮M were F1 (5⬘-TTTGCAGGCATCTGTGTGTTCCTT-3⬘, bp 702 to 725) and R1 (5⬘-CAACGCACAAGGGGCGGCCATGAC-3⬘, bp 1295 to 1272). Five microliters of the above reaction was used in a new nested RT-PCR. The new CNT1 nested oligos were F2 (5⬘-GTGTTCCTTGTCCTTCTCTTTGCT-3⬘, bp 717 to 740) and R2 (5⬘-GGCCATGACAGAGG CTGCGATTAA-3⬘, bp 1280 to 1257). The RT reactions were incubated at 94°C during 5 min to inactivate the reverse transcriptase. The PCR conditions for both reactions were 1 min, 94°C; 2 min 42°C; 3 min 72°C for 5 cycles. Then the annealing temperature was changed to 57°C and the PCR ran for another 35 cycles. For CNT3 mRNA expression, 1-step RT-PCR reactions were performed as described above using oligonucleotides derived from the published mouse sequence (15). The CNT3 primers were F (5⬘-TTGCATTTAAGATCCTGCCC-3⬘, bp 899 to 918) and R (5⬘-CCTATGAGTTTGGCGACCAT-3⬘, bp 1583 to 1564). The annealing temperature was kept to 57°C during 40 cycles. The m18S rRNA was used as control; 100 ng of total RNA

We used bone marrow-derived macrophages, which are a homogeneous population of primary and quiescent cells. Treatment with M-CSF induced proliferation of macrophages (Fig. 1A). IFN-␥ led to activation but not to proliferation. The absence of M-CSF enhances apoptosis, which progressively decreases the number of macrophages (5). Addition of IFN-␥ inhibits the M-CSFdependent proliferation. Therefore, macrophages either proliferate with M-CSF or become activated with IFN-␥ and thus stop proliferating (5). The effect of M-CSF and IFN-␥ on nucleoside incorporation into nucleic acids was monitored by 3Hthymidine and 3H-uridine incorporation into DNA and RNA, respectively. M-CSF induced the synthesis of DNA (Fig. 1B). Thymidine incorporation was not detected when IFN-␥ was added to the macrophage cultures in either the presence or absence of M-CSF (Fig. 1B). When the transcription rate was measured, both M-CSF and IFN-␥ were found to increase the amount of uridine incorporated to RNA (Fig. 1C). Characterization of nucleoside transport systems in bone marrow-derived macrophages Nucleoside transport was assessed by the uptake of uridine into actively growing cell monolayers. Total uptake was measured in the presence of external sodium. When sodium was replaced by choline, the equilibrative component was determined. Thus, the concentrative uptake is the difference between total transport and the equilibrative component. Total uptake was linear during the first 10 min and could be differentiated into two components: equilibrative and concentrative (Fig. 2A). Since a 3 min period is near initial velocity conditions, this time was used for the rest of transport experiments. To identify which nucleoside transport systems are expressed in bone marrow-derived macrophages, we analyzed the substrate specificity of the uridine uptake by cis inhibition with several nucleosides and nucleoside analogs. The concentrative uptake was partially inhibited by formycin B and almost totally by uridine (Fig. 2B). However, no inhibition was observed in the presence of NBTI. This reveals the presence of at least two concentrative systems in bone marrow: the one

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Figure 1. M-CSF induces cell proliferation and DNA and RNA synthesis, whereas IFN-␥ only induces RNA synthesis. After 18 h without M-CSF, macrophages were either kept without M-CSF (䡺) or incubated for different times in the presence of 1200 U/ml M-CSF alone (F), 300 U/ml IFN-␥ alone (E), or both (M-CSF⫹IFN-␥) (f). A) Cell counts after incubation in the three media; B) cytokine-dependent 3H-thymidine incorporation to macrophages; C) M-CSFand IFN-␥-dependent 3H-uridine incorporation to RNA Values are the mean ⫾ se of three experiments, each performed in triplicate.

inhibited by formycin B corresponds to N1; the other was probably due to N2, as reflected by the formycin Band NBTI-resistant uptake. Direct measurements of uptake with preferring substrates (guanosine, cytidine, and thymidine) in macrophages from BALB/c mice showed that concentrative transport was mostly accounted for the CNT2-type transporter, as deduced by the high guanosine uptake rates. This observation agrees with data from epithelia where CNT1 maybe

located mostly intracellularly (S. Duflot, M. Calvo, F. J. Casado, C. Enrich, M. Pastor-Anglada, unpublished results). In addition, the lack of inhibition of concentrative uridine uptake by NBTI led us to conclude that the purine-specific, guanosine-preferring, NBTI-sensitive N5 activity was absent in bone marrow macrophages. When the equilibrative component was analyzed, inhibition by NBTI reached values similar to those obtained with uridine, used as a control. This

Figure 2. Characterization of nucleoside transport systems expressed in bone marrow macrophages. A) Uridine uptake was measured in the presence or the absence of Na⫹. Total uptake is the rate of transport in the presence of sodium. Equilibrative component plus diffusion is represented by the uridine uptake in choline medium. Concentrative uptake (Na⫹-dependent) is the result of the total uptake (presence of Na⫹) minus the transport in choline medium. The result is the average of three independent measurements. B) Substrate specificity of the nucleoside uptake. Cis inhibition of uridine transport (1 ␮M) was performed in the concentrative or the equilibrative components of the uptake for 3 min. Basal represents the absence of inhibitor; formycin B and uridine were used at 100 ␮M, NBTI concentration was used at 1 ␮M. Values are the mean ⫾ se of three independent experiments, each performed in triplicate. C) mRNA expression of nucleoside transport isoforms in bone marrow macrophages and liver hepatocytes. 20 ␮g of total RNA was used in two Northern blots, each performed on independent samples. Blots were exposed for 4 days (CNT2 and ENT1) or 5– 6 wk (CNT1 and ENT2) with two intensifying screens. D) cDNA products obtained by RT-PCR from 1 ␮g of rat liver and mouse macrophages total RNA. E) Western blot analysis of CNT1 expression in various cell samples. Total cellular extracts were from several origins (rat and mouse liver, A-20 mouse B lymphocytes, and mouse bone marrow macrophages). Two independent blots were performed for each sample. 1982

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shows that the main system involved in the equilibrative uptake was es, although the presence of some low equilibrative activity due to an ei-type transport system cannot be ruled out. The specific isoforms of nucleoside transport systems were analyzed by RT-PCR, Northern, and Western blot techniques. The CNT2 mRNA coding for the system N1 was expressed in macrophages (Fig. 2C). CNT1 mRNA coding for the N2 concentrative system was not detected by Northern blot studies. However, the CNT1 gene product was found by RT-PCR (Fig. 2D). The rat CNT1 cDNA product had an expected molecular weight of 560 bp and a slightly lower faint cDNA product was obtained from mouse macrophage RNA. Furthermore, the CNT1 protein was present when a specific antibody was used in Western blot analysis (Fig. 2E). This antibody was originally generated against the rat liver protein (16), but clearly recognized the CNT1 transporter from mouse origin. Protein extracts from the mouse B cell line A20 were negative, as reported for certain human B cell lines that do not express N2 activity (13, 14). The murine CNT1 protein was slightly different from its rat counterpart. These results suggest that the CNT1 protein is expressed in macrophages and could account for the concentrative formycin B-resistant transport system N2. However, from the transport data we could not discard the presence of a very low cib-type N3 activity (broad specificity). To find out whether macrophages express the recently identified CNT3 (15), we performed RT-PCR studies. Under our conditions, a cDNA product of the specified size was obtained from NP-18 human pancreatic cell line in agreement with the expression of this gene in human pancreas (15). However, we have not been able to detect CNT3 mRNA expression in bone marrow-derived macrophages (data not shown). Finally, the gene products ENT1 and ENT2 encoding the transport activities es and ei were also present in macrophages (Fig. 2C). These data correlate the concentrative systems N1 and N2 with the nucleoside transport genes CNT2 and CNT1 and the equilibrative system es and ei with the expression of ENT1 and ENT2 genes, respectively. M-CSF regulation of nucleoside transport systems As described above, proliferating macrophages require DNA and RNA synthesis. We wanted to determine which nucleoside transport system is involved in the uptake of external nucleosides during proliferation. For these experiments, quiescence (M-CSF deprivation) or active growth (M-CSF addition) was induced in macrophages. The growth factor increased the total uptake of uridine from 17 to 30 pmol/mg prot/3 min. When each component was analyzed, N1, N2, and ei were not substantially modified by the effect of M-CSF (Fig. 3A). However, the equilibrative system es was up-regulated three- to fourfold. In agreement with transport activity data, protein expression for the concentrative system CNT1 and

mRNA expression for the concentrative CNT2 and equilibrative ENT2 systems were not affected by treatment with M-CSF (Fig. 3B, C). Furthermore, mRNA expression of the equilibrative system ENT1 was increased by ⬃threefold after 24 h of treatment (Fig. 3C). Thus, M-CSF selectively induced the equilibrative es system, probably at the transcriptional level during macrophage proliferation (Fig. 3D). Inhibition of system es blocks M-CSF-dependent proliferation and arrests macrophages in the S phase of the cell cycle To determine whether up-regulation of system es is involved in M-CSF-induced macrophage proliferation, this transport activity was blocked by incubation of macrophages with NBTI, a nucleoside analog that specifically inhibits es activity at the nanomolar range (7–10). As a result, M-CSF-induced thymidine incorporation (Fig. 4A) and proliferation were almost completely blocked, as revealed by cell counting (Fig. 4B). In contrast, M-CSF-induced uridine incorporation was not affected (Fig. 4C). Next, we analyzed the cell cycle by DAPI staining. Bone marrow macrophages growing in the presence of M-CSF showed a proliferating pattern throughout the cell cycle, as reported previously (5, 25). M-CSF starvation arrested the cell cycle at the beginning of G1. Treatment with NBTI blocked the cell cycle at the S phase (Fig. 4D). No relevant subdiploid peak was observed, ruling out that inhibition of proliferation by NBTI was due to the induction of apoptosis, as confirmed by a specific ELISA performed to analyze DNA fragmentation (data not shown). Taken together, these results indicate that up-regulation of system es by M-CSF is critical for macrophage proliferation. Effect of IFN-␥ on nucleoside transport systems in bone marrow macrophages IFN-␥ induces a series of biochemical, morphological, and functional modifications in macrophages, e.g., transcription of more than 300 genes (26). Since nucleosides are required for RNA synthesis, we wanted to determine which nucleoside transport system was altered by IFN-␥. The total nucleoside uptake in macrophages treated with IFN-␥ increased from 24 to 41 pmol uridine/mg protein/3 min. This was mainly due to the up-regulation of the concentrative systems N1 and N2 (Fig. 5). In contrast to treatment with M-CSF, the equilibrative systems es and ei remained unchanged. Similar results were found when macrophages were treated for only 4 h with IFN-␥ (data not shown). IFN-␥ not only activates macrophages, but also inhibits M-CSF-dependent proliferation, this means that IFN-␥ could inhibit the process of DNA synthesis. Therefore, we studied the effect of a 24 h treatment

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Figure 3. M-CSF specifically up-regulates the equilibrative nucleoside transport system es in bone marrow macrophages. A) Regulation of nucleoside transport activities. Cells were starved during 18 h in a MCSF-free medium, after which M-CSF was added to the medium. Transport experiments were performed after 24 h of M-CSF incubation. Uridine uptake was measured for 3 min and the transport components were analyzed based on substrate specificity. N1 is the result of the concentrative uptake inhibited by an excess of formycin B; N2, is the concentrative uptake resistant to formycin B; es activity is shown as a result of the equilibrative uptake minus the NBTI-resistant fraction; ei ⫹ D is the result of the NBTI resistant equilibrative uptake not inhibited by an excess of uridine. Values are the mean ⫾ se of four experiments, each performed in triplicate. B) Western blot analysis of CNT1 protein expressed in M-CSF treated macrophages for different periods of time. 100 ␮g of total cell lysate extracts was analyzed with CNT1 antibody. A representative Western blot of three experiments, each performed with independent samples, is shown. C) mRNA expression of CNT2, ENT1 and ENT2 in bone marrow macrophages incubated with M-CSF for various periods of time. The same filter was analyzed with CNT2, ENT1, ENT2, and 18S cDNA probes. Three filters with independent samples were analyzed. D) M-CSF differential regulation of nucleoside transport activities from cells treated for 24 h and their putative gene and protein products in macrophages. Expression values are the result of densitometric analysis performed in each blot by Phoretix software (Newcastle upon Tyne, UK).

with IFN-␥ on the nucleoside transport systems of proliferating macrophages. IFN-␥ reduced M-CSF-dependent up-regulation of the es transport activity to basal levels (Fig. 5). The pattern of regulation of CNT1, CNT2, ENT1, and ENT2 expression was similar to the corresponding transport activities. Thus, CNT1 protein levels were increased (Fig. 6A, C) between 3 and 6 h after IFN-␥ treatment. CNT2 mRNA expression rapidly increased and was maximal ⬃6 h after IFN-␥ treatment (Fig. 6B, C). ENT1 mRNA levels decreased progressively, reaching 50% of initial values 24 h after IFN-␥ treatment (Fig. 6B, C). No changes on ENT2 mRNA expression were observed (Fig. 6B, C). On the other hand, similar to what we found with CNT2 gene expression, nonquantitative CNT1 RTPCR studies suggested an increase of mRNA after 6 h of IFN-␥ treatment (Fig. 6D). Thus, actively growing macrophages showed an induced es activity, which was clearly blocked by IFN-␥. Therefore, there is a dissociation between the transport systems used when macrophages proliferate or become activated and stop proliferating. 1984

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DISCUSSION Among other metabolic and signaling functions, nucleosides are required for nucleic acid synthesis. However, certain cell types (including hematopoietic cells) such as macrophages and lymphocytes lack de novo nucleotide synthetic pathways and require exogenous nucleosides to meet their metabolic demands (7, 8). Therefore, transport across the plasma membrane is an essential regulatory first step in the nucleoside metabolism during cell growth and activation. Murine bone marrow-derived macrophages are a unique primary cultured model in which proliferation and activation can be studied separately (5, 27). M-CSF was shown to induce macrophage proliferation and DNA and RNA synthesis, whereas IFN-␥ induced only RNA synthesis and blocked proliferation. Since both macrophage proliferation and activation involve nucleic acid synthesis and need extracellular nucleosides, nucleoside transport systems and their regulation were characterized in this model. The available data on macrophage nucleoside transport are not clear. In the S1 macrophage cell line, only an equilibrative uptake has been reported (28),

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Figure 4. Inhibition of the nucleoside transport system es blocks M-CSF-dependent proliferation and arrests macrophages in the S phase of the cellular cycle. Where indicated, cells were incubated with 100 nM NBTI. Cells were processed as described in Fig. 1. A) 3H-Thymidine incorporation into DNA. B) Cell number after 24 h of MCSF incubation. C) 3H-Uridine incorporation into RNA. D) Cell cycle analysis of macrophages. Macrophages were incubated with M-CSF in the presence or in the absence of NBTI for 24 h. Then cells were washed, stained with DAPI, and fixed. The DNA content was measured using a flow cytometer. Exponentially growing cells (M-CSF) appear in the graph with two peaks corresponding to the G0/G1 phase and cells with double DNA content in G2/M phase.

whereas a poorly characterized concentrative uptake has been described in peritoneal macrophages (29, 30). However, the pattern of nucleoside transporter expression may vary according to the cell line or origin. Furthermore, no molecular data on the nucleoside transport systems were available. The present report describes for the first time that nucleoside uptake in murine bone marrow-derived macrophages is mediated through two concentrative (N1 and N2, related to CNT2 and CNT1 gene products, respectively) and two equilibrative (es and ei, corresponding to ENT1 and

Figure 5. IFN-␥ activation differentially regulates nucleoside transport systems in macrophages. Transport entities were analyzed as described in the legend for Fig. 3. Macrophages were cultured in the absence of M-CSF for 18 h. Quiescent cells were further incubated in the presence or in the absence of M-CSF and/or IFN-␥ for 24 h more. Results are the mean ⫾ se of four transport experiments, each performed in triplicate.

ENT2 genes, respectively) transport activities. The N1 and N2 concentrative activities account for ⬃ 50% of the total uptake. This study provides a characterization of the nucleoside transporters expressed in macrophages based not only on kinetics analysis, but also on mRNA or protein expression, which can be correlated with changes in the specific transport activity. We note that bone marrow-derived macrophages show differential regulation of the nucleoside transport systems in response to proliferation or activation stimuli, which may be associated with the control of specific needs for DNA and/or RNA synthesis. M-CSF-induced macrophage proliferation is linked to up-regulation of the equilibrative es transporter, but not to that of the concentrative N1 and N2 nucleoside transporters. The system es is essential to macrophage proliferation, since inhibition of this transport system by NBTI blocks DNA synthesis and M-CSF-induced macrophage proliferation. Thus, it is critical to supply nucleosides for macrophage DNA synthesis. So far, nucleoside transport regulation has been studied primarily in liver cells (31). Thus, the FAO cell line stimulated to grow by serum up-regulated the concentrative nucleoside transporter N1, whereas ENT1 seemed to be constitutively expressed (17). Moreover, during the early phase of hepatic regeneration after partial hepatectomy, concentrative systems are also increased in vivo (32). Therefore, the nucleoside transporter regulation system in operation during cell proliferation may differ from one cell type to another. In most cell types, proliferation and activation stimuli are difficult to distinguish. The liver efficiently

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Figure 6. IFN-␥ up-regulates concentrative nucleoside transport system gene products CNT1 and CNT2 and down-regulates the equilibrative ENT1 mRNA expression in macrophages incubated with M-CSF. Macrophages were cultured in the presence of M-CSF and for 24 h more in the presence of IFN-␥. A) Western blot analysis for CNT1 protein and ␤-actin, used as a loading control. B) Northern blot analysis for CNT2, ENT1, and ENT2. Murine 18S cDNA was used as a loading and transfer control. Several blots were performed at least three times on independent samples. Representative Western and Northern blots are shown. C) Quantification analysis of the gene product expression by Phoretix software. D) RT-PCR of CNT1 in murine bone marrow-derived macrophages. Growing cells in the presence of M-CSF were treated with IFN-␥ for 6 h and RNA was isolated. A representative preparative 1% agarose gel is shown.

extracts afferent nucleosides from the blood, but it also shows a high endogenous capacity for nucleoside biosynthesis (33). Other regulatory steps in nucleic acid synthesis pathways cannot be ruled out. In the GN4 rat liver epithelial cell, the first steps in the de novo synthesis of uridine nucleotides, catalyzed by carbamoyl phosphate synthetase (CPSII), are regulated during proliferation and CPSII activity is associated with increased growth in normal and tumor cell lines (34). Moreover, in the hepatoma cell line HepG2, the ENT1 transporter is constitutively overexpressed and so es is the main system responsible for nucleoside uptake, in contrast to normal hepatocytes (17, 31). Thus, contrary to dedifferentiated cell lines, a nontransformed model may require fine-tuning of the equilibrative transporter. Here we show that macrophage activation by IFN-␥ increases concentrative transporters. Since IFN-␥ macrophage activation induces only RNA synthesis, the nucleosides for RNA synthesis may be transported mainly by Na⫹-dependent agencies in macrophages. Up-regulation of concentrative systems by activating stimuli may characterize immune system cells. Thus, activation of the human Raji B lymphocytes by LPS or PMA up-regulates the concentrative systems (13, 14). Inhibition of the proliferation by IFN-␥ down-regulates the system es. Therefore, IFN-␥ blocks a signal induced by M-CSF that is needed for cell proliferation. This may be one of the mechanisms by which IFN-␥ hinders proliferation, although other actions are also involved. IFN-␥ may also induce expression of the cdk inhibitors p21waf and p27kip and thus stop the cell cycle 1986

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at the G1/S interphase (5), whereas inhibition of the system es stops the cell cycle at the S phase because of the lack of nucleosides for DNA synthesis. This study indicates that although the nucleoside requirements for nucleic acid synthesis during activation and proliferation may be similar, the nucleoside transport pathways involved are distinct. The signal transduction generated by IFN-␥ is mediated through the JAK/STAT pathway (35). However, to induce proliferation, M-CSF requires activation of the Raf/MEK/ ERK cascade (27, 36), which stimulates mitogenic agents such as c-jun, c-fos, and c-Myc. The latter is necessary for M-CSF-induced mitogenesis (37). M-CSFdependent proliferation is probably blocked by the inhibition of c-Myc expression by IFN-␥ (5). Although the mechanism by which IFN-␥ blocks the equilibrative nucleoside transport system induced by M-CSF is unknown, the role of c-Myc repression should be evaluated. Our results may be of physiological and pharmacological interest. Macrophages play a key role at the inflammatory loci, where they arrive 24 to 48 h after the lesion and remain until inflammation disappears (38), i.e., while stimulated Th1 cells produce IFN-␥. However, the persistence of macrophages at the inflammatory loci is related to the pathogenesis of a wide range of inflammatory diseases (39). This report is important not only to understand how macrophages are able to differentiate the nucleoside salvage pathways used for DNA or RNA synthesis requirements, but could also provide new insights for the treatment of chronic inflammatory diseases through the inhibition of nucle-

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oside transport. The authors thank Dr. I. Fabregat for supplying the m18S cDNA. This study was supported by grants SAF99 – 0115 and 2FD97–1268 to M.P.A., SAF98 –102 and PM98/0200 to A.C., and a grant from the University of Barcelona to A.F. J.G.M. is the recipient of a research fellowship from Programa BEFI (Fondo de Investigacio´n Sanitaria, Instituto de Salud Carlos III, Spain). R.V. holds a F.P.I. fellowship (Universitat de Barcelona). J.X. holds a fellowship from the Comissio´ Interdepartamental de Recerca i Innovacio´ Tecnolo`gica. M.C. is the recipient of a fellowship from the Fundacio´ August Pi i Sunyer.

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Received for publication January 11, 2001. Revised for publication May 21, 2001.

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