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,1,12
* Departament de Fisiologia (Biologia del Macròfag) and Fundació August Pi i Sunyer, Campus de Bellvitge; and
Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, E-08028 Barcelona, Spain
2Correspondence: Laboratori de Fisiologia Molecular, Departament de Bioquímica i Biologia Molecular, Universitat de Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain. E-mail: afelipe{at}porthos.bio.ub.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(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., García-Manteiga, J., Valdés, 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.
Key Words: nucleoside uptake • interferon • macrophage colony-stimulating factor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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), 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
8
9
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
8
9
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
13
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
8
9
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 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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 ß-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 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 µM of
3H-nucleoside (1 µCi/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
(5x104) 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 µCi/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 µg 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 20xSSC (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.
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 µg/ml aprotinin, 1 µg/ml
leupeptin, 86 µg/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 µg) 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)
.
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 µg/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 µg 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
µl 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 µM 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 µM 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 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'-CGCAGAATTCCCACTCCCGACCC-3', bp 482 to 498) and R
(5'-CCCAAGATCCAACTACGAGC-3', bp 694 to 675).
Ten µl 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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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-CSF-dependent 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 3H-thymidine 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 inhibited by formycin B corresponds to N1; the
other was probably due to N2, as reflected by the formycin B- and
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 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
, 3C
). 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
8
9
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 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 RT-PCR
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.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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)
, 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 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 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 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-CSF-dependent 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 nucleoside transport.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 11, 2001.
Revision received May 21, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in human B-lymphocytes. J. Biol. Chem. 273,26939-26945. Blood 95,3823-3831. Annu. Rev. Immunol. 15,749-795[Medline]
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) or incubated
for different times in the presence of 1200 U/ml M-CSF alone (•), 300
U/ml IFN-
), or both (M-CSF+IFN-
).
A) Cell counts after incubation in the three media;
B) cytokine-dependent 3H-thymidine
incorporation to macrophages; C) M-CSF- and
IFN-
