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Volume 17, Number 14,
Issue of July 15, 1997
pp. 5305-5315
Copyright ©1997 Society for Neuroscience
Neurons Promote Macrophage Proliferation by Producing
Transforming Growth Factor- 2
Alexandre Dobbertin1,
Peter Schmid2,
Michèle Gelman1,
Jacques Glowinski1, and
Michel Mallat1
1 Institut National de la Santé et de la
Recherche Médicale U 114, Chaire de Neuropharmacologie,
Collège de France, 75231 Paris Cedex 05, France, and
2 Ciba-Geigy Limited, CH-4002 Basel, Switzerland
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The infiltration of bone marrow-derived macrophages into the CNS
contributes to growth and reactions of microglia during development or
after brain injury. The proliferation of microglial cells is stimulated
by colony-stimulating factor 1 (CSF-1), an astrocyte-produced growth
factor that acts on mononuclear phagocytes. In the present study, we
have shown, using an in vitro model system, that rodent neurons obtained from the developing cerebral cortex produce a soluble
factor that strongly enhances the proliferation of macrophages cultured
in the presence of CSF-1. Both macrophages isolated from the developing
brain and those from the adult bone marrow were stimulated. Kinetic
analyses of [3H]thymidine incorporation into
macrophages indicated that their response to the neuron-derived factor
involved a shortening of the cycle of proliferating cells. The effect
of neurons on macrophages was blocked in the presence of antibodies
neutralizing transforming growth factor- 2 (TGF-2), whereas
recombinant TGF-2 stimulated macrophage proliferation in the
presence of CSF-1. Neuronal secretion of TGF-2 was confirmed by
reverse transcription-PCR detection of TGF-2 transcripts and
immunodetection of the protein within neurons and in their culture
medium. In situ hybridization and immunohistochemical
experiments showed neuronal expression of TGF-2 in sections of
cerebral cortex obtained from 6-d-old rats, an age at which extensive
developmental recruitment of macrophages occurs in this cerebral
region. Altogether, our results provide direct evidence that neurons
have the capacity to promote brain macrophage proliferation and
demonstrate the role of TGF-2 in this neuronal function.
Key words:
microglia;
bone marrow-derived macrophage;
neuron;
colony-stimulating factor 1;
transforming growth factor-;
rat
INTRODUCTION
The infiltration of the CNS by bone marrow-derived
monocytes contributes to the establishment and turnover of microglia.
In the developing CNS, invading monocytes proliferate and are
transformed into ameboid microglia, also called brain macrophages (BMs)
(Ling and Wong, 1993 ). Maturation of the CNS is associated with a
progressive differentiation of BMs into resting, ramified microglia.
However, several pathological states lead to the reappearance of
proliferating BMs as a consequence of increased infiltration of
monocytes or activation and transformation of ramified microglial cells
into BMs (Perry et al., 1994). Accumulating evidence indicates that BMs
produce trophic as well as toxic compounds that can act on different
CNS lineages and promote inflammatory reactions by presenting antigen
to T-cells (Mallat and Chamak, 1994; Gehrmann et al., 1995). Hence, the
regulation of intracerebral proliferation of macrophages is a key issue
in the understanding of physiological and pathophysiological remodeling
of CNS tissue.
As with other tissue macrophages or their precursors, the proliferation
of BMs can be stimulated by colony-stimulating factors (CSFs), such as
CSF-1 (also called macrophage CSF) and granulocyte macrophage CSF
(GM-CSF), which are encoded by distinct genes (Giulian and Ingeman,
1988; Metcalf, 1989; Hao et al., 1990; Roth and Stanley, 1992;
Théry and Mallat, 1993). The expression of CSF-1 and CSF-1 mRNA
has been detected in extracts of developing and adult rodent CNS
(Théry et al., 1990, Hulkower et al., 1993, Chang et al., 1994,
Roth and Stanley, 1996). Recent analyses of mice genetically deficient
in CSF-1 indicate a primary role of CSF-1 in the recruitment of BMs.
Indeed, despite the presence of resting, ramified microglia, the
proliferation of activated microglia and the occurrence of BM
phenotypes appeared dramatically reduced in the mutant brain after
ischemic or mechanical injury (Raivich et al., 1994; Berezovskaya et
al., 1995).
Cellular sources of CSF-1 were studied in cultures derived from
human or rodent CNS; astrocytes were found to be the main source and to
produce this factor constitutively (Hao et al., 1990; Frei et al.,
1992; Théry et al., 1992; Lee et al., 1993; Théry and
Mallat, 1993). In addition, astrocytes stimulated with inflammatory
cytokines synthesized GM-CSF (Malipiero et al., 1990; Aloisi et al.,
1992), indicating that these cells can promote macrophage growth by
producing at least two different mitogenic agents. In contrast, the
influence of neurons on macrophage proliferation is poorly defined.
CSF-1 has been detected in neuronal cultures derived from mouse
cerebellum but not in those obtained from cerebral cortex (Théry
et al., 1990; Nohava et al., 1992). This prompted us to set up an
in vitro coculture system to study the influence of neurons
on the growth of macrophages isolated from bone marrow or from CNS. We
observed that rat neurons stimulated the proliferation of macrophages
by producing a factor enhancing the activity of CSF-1. This
neuron-derived factor was identified as a member of the transforming
growth factor- (TGF-) family (Massagué et al., 1994),
TGF-2.
MATERIALS AND METHODS
Reagents for cell cultures
Recombinant human CSF-1 (rhCSF-1; specific activity,
2-5 × 106 U/mg protein), rhTGF-1,
rhTGF-3, and purified goat polyclonal antibodies neutralizing
TGF-2, TGF-3, interleukin 3 (IL-3), GM-CSF, or tumor necrosis
factor- (TNF-) were obtained from R & D Systems (Abingdon, UK);
rhTGF-2, mouse monoclonal antibodies (mAbs) neutralizing TGF-1,
-2, and -3 (IgG1), TGF-2 and -3 (IgG2b), or IL-7 came from
Genzyme (Le Perray en Yvelines, France). Affinity-purified rabbit
polyclonal anti-TGF-2 antibodies raised against a peptide fragment
of mature human TGF-2 (residues 50-75) were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Methyl-[3H]thymidine (specific activity, 46 Ci/mmmol) was purchased from Amersham (Les Ulis, France). Insulin,
iron-free transferrin, progesterone, putrescin, and selenium came from
Sigma (L'isle d'Abeau, France). All other components of the culture
media were from Life Technology (Eragny, France). Contamination of the
culture media with lipopolysaccharide was <0.01 ng/ml, as indicated by
the limulus amebocyte assay from Sigma. Nonpurified murine
CSF-1 was obtained as a conditioned medium from L929 cells cultured at
2 × 105 cells/ml for 7 d in DMEM
supplemented with 10% fetal calf serum (FCS) (Stanley and Heard,
1977). All cell types were cultured in plastic dishes from Nunc
(Naperville, IL).
Cell populations
Bone marrow-derived macrophages. Bone marrow-derived
macrophages (BMMs) were obtained from cultures of bone marrow collected from male Sprague Dawley rat femurs (Charles River, St. Aubin les
Elbeuf, France) as described by Tushinski et al. (1982). Bone marrow
cells were flushed out from the femur with a 25 gauge needle into
ice-cold PBS containing 33 mM glucose. The marrow plugs
were dispersed and incubated for 10 min at 4°C in Gey's solution to lyse the red cells and then washed in DMEM (250 gm, 10 min at 4°C).
Cells were seeded into Petri dishes (1.5 × 106
cells/ml) in 10 ml of DMEM supplemented with 10% FCS and 20% L929-conditioned medium (L9CM). After a 3 d incubation (37°C, 5% CO2), the culture medium containing the
nonadherent cells was transferred into new plates, and 5 ml of fresh
complete medium was added. After 3 d of culture, adherent cells
were discarded, whereas nonadherent cells were collected and washed
twice before being used as BMMs in the experiments. The purity of
isolated BMMs was checked by immunocytochemical detection of specific
macrophage markers. Virtually all the cells bore ED1 macrophage antigen
as well as complement receptor type 3.
Brain macrophages. BMs (>99% pure) were isolated from
2-week-old primary glial cultures derived from the cerebral cortex and striatum of embryonic day 17 (E17) OFA rats (IFFA Credo) and grown in
DMEM supplemented with 10% FCS, as described previously (Théry et al., 1991). Cells were washed in DMEM before use.
Neurons. Neuronal primary cultures were derived from E17 rat
cerebral cortex or from E14 rat mesencephalon as described previously by Théry et al. (1991). Briefly, dissociated cells were seeded on
plastic or on 14 mm glass coverslips coated with polyornithine (15 µg/ml; 4 × 105 cells/glass coverslip) and
cultured in a chemically defined medium (CDM) consisting of DMEM/F12
nutrients (1:1) supplemented with 33 mM
D-glucose, 2 mM L-glutamine, 9 mM NaHCO3, 5 mM HEPES, 25 µg/ml insulin, 100 µg/ml iron-free transferrin, 20 nM
progesterone, 60 µM putrescin, and 30 nM
selenium. In these culture conditions, >95% of the cells bore
neuronal markers such as microtubule-associated protein 2 (MAP2) and
neurofilament subunits, whereas <5% of the cells expressed glial
fibrillary acidic protein (GFAP) or macrophagic ED1 antigen. Neurons
from cerebellum were obtained from 6-d-old postnatal OFA rats as
described by Van Vliet et al. (1989) and cultured in CDM supplemented
with 25 mM K+.
Coculture system
The influence of neurons on macrophages was studied in coculture
allowing exchange of soluble factors between the two cell populations.
Macrophages were seeded at low density into 16 mm wells (8000-20,000
cells/well) in 600 µl of CDM (described above) supplemented with 20%
L9CM or 2% FCS and rhCSF-1. Twenty-four hours after macrophage
seeding, neurons cultured on top of 14 mm glass coverslips were
introduced with their glass substrate into the wells. Glass coverslips
were mounted on 3 mm paraffin feet allowing space between the
macrophages and the cell free bottom sides of the slides introduced
into the wells. Controls were performed by adding glass coverslips
devoid of cells in wells containing the macrophages. At the end of the
coculture, the glass coverslips were removed, and the macrophages were
fixed for immunocytochemical analysis and cell count. The lack of any
contaminating cells derived from the neuronal compartment in the bottom
of the wells was checked by introducing glass coverslips coated with
neurons in wells devoid of macrophages.
Cell number determination
For routine cell counting macrophages were fixed by a 10 min
incubation at 4°C in 2.5% glutaraldehyde, which was added to the 16 mm wells without removing the culture medium. Cells were then washed
extensively in PBS and incubated 10-15 min with 0.05% toluidine blue
in 2% Na2CO3. Stained cells were washed with
distilled water, air dried, and examined with a Nikon optical
microscope. Cell growth was determined in each well by counting the
number of macrophages in 15 high-power fields (magnification, 200×)
covering 1/40 of the surface of the well. The percentage of cells
recovered 24 hr after plating was estimated from the number of cells
counted (mean from four sister wells) using the formula: percentage of cells = 100 × (number of cells counted × 40/number of
cells plated).
[3H]Thymidine incorporation
and autoradiography
Cultured BMMs were incubated for different times in the presence
of 1 µCi/ml methyl-[3H]thymidine. At the end of
the labeling period, BMMs were fixed with 2.5% glutaraldehyde (10 min
at 4°C), and the wells were washed six times with PBS before air
drying. The wells were then coated with K5 emulsion in gel form (Ilford
Scientific Products) and stored at 4°C for 3 d before
developing. Cultures were then counterstained with 0.05% toluidine
blue in 2% Na2CO3, and both
[3H]thymidine-labeled and unlabeled cells were
counted under an optical microscope as described above.
ELISA detection of TGF-2
A commercially available immunoassay (ELISA) set up to detect
biologically active human TGF-2 (R & D Systems) was used to quantify
this cytokine in the medium of cultures according to the instructions
of the manufacturer. Latent TGF-2 was also assayed after activation
by transient acidification of the culture media: pH 2 for 20 min by
adding 100 mM HCl to the media, followed by neutralization
with an equal amount of NaOH and addition of 20 mM HEPES.
Controls were performed using the culture medium incubated free of
cells.
Reverse transcription-PCR and Southern blot procedure
Total RNA was isolated from cells lysed in guanidinium
isothiocyanate as described by Chomczynski and Sacchi (1987). Two
micrograms of RNA were reverse-transcribed (avian myeloblastosis virus,
42°C) using a kit from Promega (Charbonnières, France) and
TGF-1-3 cDNAs were amplified by PCR (30 cycles) using specific
primers and the following conditions for each cycle: 30 sec at 94°C,
1 min at the annealing temperature (58°C for TGF-1, 62°C for
TGF-2, and 60°C for TGF-3), and 1 min elongation at 72°C. The
final extension was allowed to continue for 10 min. Amplified products were size-fractioned by electrophoresis through a 1.5% agarose gel.
Gels were then denatured for 30 min in a solution of 0.5 M
NaOH and 1.5 M NaCl, neutralized for 20 min in 0.5 M Tris buffer, pH 8, containing 1.5 M NaCl, and
blotted onto nitrocellulose filters (Bioprobe Systems,
Montreuil-sous-Bois, France). Filters were hybridized overnight at
65°C with 32P-labeled cDNA probes in 5× SSC, 50 mM PBS, pH 6.5, 5× Denhardt's solution, 100 µg/ml
salmon sperm DNA, and 0.4% SDS. Blots were washed at 65°C in 1× SSC
and 0.1% SDS.
Rat TGF-1 sense and antisense primers were
5 -GAAGTCACCCGCGTGCTAAT-3 and 5-TTGCGACCCACGTAGTAGAC-3, giving
a product of 800 bp (Qian et al., 1990); mouse TGF-2 sense and
antisense primers were 5-CTCCTGCATCTGGTCCCGGT-3 and
5-GCACAGCGTCTGTCACGTCG-3, giving a product of 592 bp (Proetzel et al.
1995); and mouse TGF-3 sense and antisense primers were
5-GAAGATGACCATGGCCGTGG-3 and 5-GCTGGCCTCAGCTGCACTTA-3, giving
a product of 470 bp (Proetzel et al., 1995). The TGF-1 probe was
a 730 bp SacI-PvuII fragment of porcine TGF-1
cDNA (Kondaiah et al., 1988). The TGF-2 probe was a 2.35 kb simian
cDNA (Hanks et al., 1988). For the TGF-3 probe, a 339 bp
NcoI-SalI fragment of murine TGF-3 cDNA
(Schmid et al., 1991) was used. Positive controls for TGF-
expression were provided with cDNA obtained from adult mouse brain, rat
epithelial liver cells (REL cell line), and peripheral nerve ganglions
from adult rat (Unsicker et al., 1991; Mercier et al., 1995). PCR
controls performed with the non-reverse-transcribed RNA samples were
negative.
Immunocytochemistry
Immunoperoxidase detection of neuronal, astrocytic, or
macrophagic markers in cultures was performed according to a procedure described previously (Chamak et al., 1994). Primary mAbs applied for 1 hr at room temperature were 1/300 IgG1 anti-MAP2 from BioMakor (Rehovot, Israel), 1/1000 SMI 31 IgG1 anti-phosphorylated neurofilament H from Sternberger Monoclonal Inc. (Baltimore, Maryland), 1/200 IgG1
anti-GFAP, from Amersham, and 1/100 IgG1 ED1 or 1/100 IgG2a OX42
anti-complement receptor type 3 from Serotec (Bicester, UK). mAbs were
detected by peroxidase-conjugated goat anti-mouse IgG antibodies from
Byosis (Compiègne, France) or by the biotin-avidin-peroxidase method using the Vectastain Elite kit from Vector Laboratories (Peterborough, UK) and diaminobenzidine hydrochloride (Sigma) as the
chromogen.
For double immunofluorescent staining, fixed cells (4%
paraformaldehyde) permeabilized with 0.1% Triton X-100 (Chamak et al., 1994) were saturated with 20% normal donkey serum (Jackson
ImmunoResearch, West Grove, PA) diluted in PBS for 1 hr at room
temperature. Cells were then incubated in PBS containing 2% normal
donkey serum (1 hr at room temperature followed by washes) with
sequentially 1/2000 rabbit polyclonal anti-TGF-2 (Santa Cruz
Biotechnology), 1/100 fluorescein isothiocyanate (FITC)-conjugated
donkey F(ab)2 anti-rabbit IgG (Jackson ImmunoResearch), and 1/300
anti-MAP2 mAb and 1/100 tetramethyl rhodamine isothiocyanate
(TRITC)-conjugated donkey F(ab)2 anti-mouse IgG.
For in situ detection of TGF-2 and MAP2, postnatal day 6 (P6) rats were deeply anesthetized with diethyl ether and then perfused transcardially with PBS followed by 2%
periodate-lysine-paraformaldehyde fixative containing 2%
paraformaldehyde. Brains were post-fixed in the same fixative (2 hr at
4°C), cryoprotected by overnight (4°C) immersion in 10% sucrose in
PBS, and rapidly frozen. Cryostat cut coronal sections (20 µm thick)
were mounted onto poly-L-lysine-coated slides, air dried,
and stored at  20°C before use. Double immunocytochemical detection
of TGF-2 and MAP2 was performed as described above with minor
modifications; incubations and washes were all performed in the
presence of 0.1% Triton X-100, and anti-TGF-2 antibodies were
applied overnight at 4°C.
Controls were performed by substituting primary mAbs with unrelated
mouse IgG1 or IgG2a and by incubating anti-TGF-2 polyclonal antibodies with a 20-fold excess of the synthetic peptides used to
generate the antisera (supplied by Santa Cruz Biotechnology).
In situ hybridization
Sample preparation and hybridization were performed according to
a reported procedure (Gautron et al., 1992). Frozen tissue sections
fixed as described above were thawed, dehydrated in ethanol, and air
dried before post-fixation in 2% paraformaldehyde for 20 min at room
temperature and washes in PBS. Samples were treated for 8 min at room
temperature with 20 µg/ml proteinase K (Boehringer Mannheim,
Mannheim, Germany) diluted in 50 mM Tris and 5 mM EDTA, pH 8, washed in PBS, fixed again with 2%
paraformaldehyde (5 min, room temperature), and rinsed in PBS and
H2O. Sections were then acetylated, washed, and dehydrated
with solutions of increasing ethanol concentration before air
drying.
Sense and antisense TGF-2 RNA probes were labeled with
35S-uridine triphosphate (UTP, >1000 Ci/mmol; Amersham) to
a specific activity of >109 cpm/µg using
SP6 or T7 RNA polymerase according to the instructions of the
manufacturer (Stratagene, La Jolla, CA). The riboprobe templates were
339-nucleotide-long fragments, subcloned into pGEM5 (Promega), and
corresponded to the cDNA sequence encoding the mature form (including
the stop codon) of murine TGF-2 (Schmid et al., 1991). Labeled
probes were fragmented by alkaline digestion. Hybridization was
performed overnight at 52°C in 50% formamide, 10% dextran sulfate,
0.3 M NaCl, 20 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 5 mM EDTA, 1× Denhardt's
reagent, and 0.5 mg/ml yeast tRNA (Sigma) supplemented with a 5 × 104 cpm/µl 35S-UTP-labeled RNA
probe. Slides were washed as follows: 5× SSC and 10 mM
dithiothreitol (DTT), 30 min at 50°C; 2× SSC, 50% formamide, and 10 mM DTT, 20 min at 60°C; and twice in washing solution
containing 0.3 M NaCl, 20 mM Tris, and 5 mM EDTA, 10 min at 37°C. Sections were treated with 20 µg/ml RNase A and 2 µg/ml RNase T1 (Boehringer Mannheim) in the
washing solution for 30 min at 37°C, followed by washes in washing
solution (5 min, 37°C), 2× SSC, and finally 0,1 × SSC (15 min,
37°C each). Slides were dehydrated, air dried, and coated with
Ilford K5 photo emulsion before exposure for 2 weeks at 4°C.
After development, slides were dehydrated and stained with 0.05%
toluidine blue.
RESULTS
Influence of CSF-1 on the growth of cultured BMMs
Macrophages directly harvested from bone marrow culture (BMMs)
provide a highly homogeneous cell population and are much easier to
obtain than circulating monocytes from rodent blood. Therefore, BMMs
were used to investigate the early influence of neurons on mononuclear
phagocytes that infiltrate the CNS parenchyma. Consistent with previous
reports (Tushinski et al., 1982; Tushinski and Stanley, 1985), in
vitro growth of isolated BMMs required addition of CSF-1 to the
culture medium (Fig. 1). The concentration of CSF-1 for optimal stimulation has been shown to depend on the macrophage density
in cultures, because binding of CSF-1 to BMMs leads to internalization
and degradation of the cytokine (Tushinski et al., 1982). In our assay,
using a low cell density, growth of BMMs estimated 4 d after cell
plating was found to be optimal at a rhCSF-1 concentration of 2 ng/ml.
Similar growth was obtained using 20% L9CM as a source for CSF-1 (Fig.
1). In these optimal conditions, quantitation of BMMs 1 d after
plating showed that according to the experiments, a proportion of
15-50% of the cells seeded was rescued from death, and these cells
were adhering to the plastic substrate. Figure 2 shows
that beyond the first day after BMM isolation, surviving cells
stimulated with CSF-1 underwent proliferation, as indicated by the
progressive increase in culture well BMM number. In cultures without
CSF-1, a drop in BMM number was already obvious between 2 and 24 hr
after cell isolation, indicating that the survival of freshly harvested
BMMs was strongly dependent on the presence of CSF-1 (Fig. 2).
Fig. 1.
Influence of neurons and CSF-1 on BMM growth. BMMs
were seeded at a density of 10,000 cells/well in CDM supplemented with L9CM or rhCSF-1 and 2% FCS. Cells were cultured for 96 hr with neurons
(hatched bars) or without neurons (open
bars) added for the last 72 hr. At the end of the culture, the
number of BMM was determined by counts in 15 fields for each well as
described in Materials and Methods. Each value is the mean number of
cells ± SEM from four wells in one experiment representative of
three independent experiments. Comparison between numbers of BMMs in the presence and absence of neurons was performed for each CSF-1 concentration using Student's t test
(*p < 0.05).
[View Larger Version of this Image (26K GIF file)]
Fig. 2.
Effect of CSF-1 and TGF-2 on the time course of
BMM growth. BMMs were seeded at a density of 10,000 cells/well in CDM
supplemented with 2% FCS with or without 10 ng/ml rhCSF-1 or 0.1 ng/ml
rhTGF-2. The number of BMMs was determined at different times after
cell plating. Values are mean number of cells ± SEM from four
wells in one experiment representative of two independent experiments. Open circles, Culture without recombinant cytokines;
filled squares, culture with rhCSF-1; open
triangles, culture with rhTGF-2. In the presence of CSF-1,
the estimated proportion of BMM recovered 24 hr after cell plating was
50%; from this time point, the number of BMMs increased significantly
within 24 hr (p < 0.05). Statistical analyses were performed by one-way ANOVA followed by Dunnett's test.
[View Larger Version of this Image (15K GIF file)]
Influence of CNS neurons on BMM proliferation
The paracrine effect of neurons on BMM growth was investigated in
a coculture system that allowed the exposure of BMMs to soluble factors
released by neurons. Figure 1 illustrates that in the absence of CSF-1,
the addition of cultured neurons (1 d in vitro) from E17
cerebral cortex to wells seeded with BMMs failed to rescue BMMs from
degeneration. This result is consistent with the lack of CSF-1
production by these neurons (Théry et al., 1990) (A. Dobbertin
and M. Mallat, unpublished results). However, at optimal concentration
of CSF-1 (2 or 10 ng/ml rhCSF-1 or 20% L9CM), the presence of neurons
led to a twofold increase in the number of BMMs after 3 d of
coculture (Fig. 1). Increased BMM numbers were observed when starting
the cocultures with highly pure neuronal populations grown on glass
coverslips in CDM. The purity of the neuronal population was also
checked at the end of the coculture. More than 95% of the cells
displayed the typical shape of cultured neurons and expressed specific
neuronal markers such as MAP2 or the 200 kDa neurofilament subunit.
Immunocytochemical detection of the ED1 antigen and GFAP confirmed that
contaminating brain macrophages and astrocytes within the neuronal
culture remained <5% of the total cell population after a 3 d
exposure to low FCS (2%) and rhCSF-1 or L9CM.
The number of BMMs was also significantly increased, although to a
lower level, when medium conditioned by neurons was added to BMM
culture wells instead of living neurons. Thus, neurons do not require
stimulation by BMMs to release growth factors acting on the macrophages
(data not shown).
These results indicate that neuron-derived factors are ineffective by
themselves to promote macrophage survival and proliferation but enhance
the mitogenic activity of CSF-1. This enhancement of proliferation was
further characterized by kinetic analyses of DNA synthesis using BMMs
that had entered a quiescent state before exposure to neurons. It has
been shown that removal of CSF-1 from the culture induces proliferating
BMMs to enter a quiescent (G0/G1)
state and that the cells can reenter the growth cycle if CSF-1 is
re-added soon enough to avoid BMM death (Tushinski and Stanley, 1985).
In our model, preliminary studies monitoring cell death and DNA
synthesis showed that BMM quiescence was fully achieved after a 24 hr
deprivation of CSF-1. Kinetics of [3H]thymidine
nuclear incorporation indicated that these BMMs were able to synthesize
DNA after re-addition of CSF-1 (Fig. 3). During the
first 20 hr, the number of BMMs entering the S phase of the cell cycle
was significantly increased when the cells were stimulated with both
neurons and CSF-1 compared with CSF-1 alone. However, after 24 hr, the
same proportion of cells (70%) was labeled under both conditions,
indicating that neurons act by shortening the cell cycle of BMMs
stimulated by CSF-1 rather than by inducing proliferation of BMMs
unresponsive to CSF-1 alone (Fig. 3).
Fig. 3.
Effects of neurons and CSF-1 on the time course of
DNA synthesis in quiescent BMMs. Isolated BMMs were cultured for 2 d in the presence of CSF-1 (20% L9CM) and washed, and synchronization of the cultures in a growth-arrested phase was obtained by a 24 hr
incubation in CDM free of CSF-1. Cells were then incubated in CDM
supplemented with L9CM and 1 µCi/ml
[3H]thymidine with or without neurons. At
indicated times, [3H]thymidine incorporation was
stopped, and macrophages were processed for autoradiography. Results
are from one experiment representative of three independent
experiments. Values are presented as percentage of labeled nuclei
(mean ± SEM) determined in four wells for each condition.
Filled squares, Percentage of labeled macrophage nuclei after coculture with neurons; open circles, control
without neurons. Comparison of the proportions of labeled BMMs cultured
with or without neurons was performed at different times using
Student's t test (16 hr, p = 0.0002; 20 hr, p = 0.06).
[View Larger Version of this Image (12K GIF file)]
Stimulation of BMM proliferation in the presence of CSF-1 was also
observed using neuronal cultures derived from E17 cerebral cortex, E14
mesencephalon, or P6 cerebellum (Fig. 4). Comparison between neurons derived from E17 cortex and grown in CDM for 1 or
7 d before coculture indicated that the in vitro
neuronal maturation did not alter their ability to increase BMM
proliferation (Fig. 4). Similar results were obtained using
mouse-derived BMM and neurons from embryonic mouse cerebral cortex or
mesencephalon (data not shown).
Fig. 4.
Effect of different neuronal populations on BMM
growth. Neurons from different CNS regions were seeded on glass
coverslips (4 × 105 cells/coverslip) and grown
for different times before coculture with BMM in CDM supplemented with
20% L9CM. Cx 1DIV, Cx 7DIV, Neurons from E17 cerebral
cortex 1 or 7 d in vitro before coculture; Mes, mesencephalic neurons from E14 rat embryos 1 d
in vitro before coculture; Cb, P6
cerebellar neurons 3 d in vitro before coculture. The numbers of BMMs counted after 72 hr of coculture are expressed as
percentages of the mean control value set as 100% (BMMs in wells
without added neurons). Each value is the mean ± SEM from counts
in four wells in one experiment representative of three independent
experiments. The actual number of BMMs counted in control conditions
was 126 ± 23. Coculture and control values were compared using
Student's t test (*p < 0.01).
[View Larger Version of this Image (23K GIF file)]
TGF-2 is responsible for the increased proliferation of
macrophages in the presence of neurons
The possible involvement of cytokines suggested to modulate CSF-1
activity (Chen et al., 1988; Celada and Maki, 1992; Guilbert et al.,
1993; Jacobsen et al., 1994) was investigated in our model by adding
different blocking antibodies to the coculture medium. As shown in
Figure 5A, neuronal stimulation was not
significantly affected in the presence of antibodies neutralizing
GM-CSF, IL-3, TNF-, or IL-7. In contrast, the effect of neurons on
BMM proliferation was fully prevented in the presence of two different
monoclonal antibodies directed against TGF- and neutralizing three
(1-3) or two (2 and 3) of the TGF- isoforms (Fig.
5A). A primary role for TGF-2 in BMM proliferation was
further demonstrated by the complete blockade of the neuronal
stimulation with monospecific antibodies directed against whole
TGF-2 (goat antibodies from R & D Systems) or a TGF-2 fragment
(rabbit antibodies from Santa Cruz Biotechnology). In contrast,
anti-TGF-3 antibodies did not affect stimulation of BMMs by neurons
(Fig. 5A) when used at a concentration neutralizing up to
0.1 ng/ml rhTGF-3.
Fig. 5.
Neutralization of neuronal stimulation by
different anti-TGF- antibodies. Cocultures were all performed in CDM
supplemented with 2% FCS and 10 ng/ml rhCSF-1. Antibodies were added
together with neurons into wells previously seeded with BMM
(A) or BM (B). Final
concentrations of added antibodies were 20 µg/ml for polyclonal anti-TNF- antibodies, anti-IL7 mAb, anti-TGF-2 and -3 mAbs, and control unrelated IgG2b or 10 µg/ml for other mAbs or polyclonal antibodies. For each antibody, the results of one experiment
representative of at least two independent experiments are shown.
Hatched bar, Control number (set as 100%) of BMMs or
BMs cultured without neurons. Open bar, Number
(percentage of control) of BMMs (A) or BMs
(B) counted at the end of 72 hr cocultures in the
presence or absence of antibodies. Values are mean ± SEM from
counts in four wells. The actual numbers of cells in the seven
(A) and two (B) controls were A, 68 ± 10, 222 ± 14, 104 ± 7, 42 ± 2, 420 ± 12, 64 ± 2, 308 ± 20;
B, 123 ± 10 and 63 ± 8 (from
top to bottom). Neuronal stimulation
without antibodies compared with control values (without neurons) were
all significant (p < 0.01;
p < 0.05 for the experiment performed with
anti-TGF-2 and -3 antibodies). Addition of unrelated mAbs, rabbit
IgGs, or monospecific antibodies directed against GM-CSF, IL-3, IL-7,
TNF-, or TGF-3 did not significantly alter neuronal stimulation.
Comparisons between anti-TGF- antibodies (mAbs neutralizing two or
three isoforms or rabbit monospecific anti-TGF-2) and unrelated IgGs
of matched isotype or species confirmed directly the specificity of
blocking effects (in all cases p < 0.05).
Significant differences were also checked between goat monospecific
anti-TGF-2 and anti-TGF-3 in cocultures with BMMs
(p < 0.05) or BMs
(p < 0.01). Statistical analyses were
performed using one-way ANOVA followed by multiple comparison
Bonferroni's test.
[View Larger Version of this Image (26K GIF file)]
The above-mentioned effects were observed with cultured macrophages
derived from bone marrow, used as a model for phagocyte precursors
infiltrating the CNS. In another series of experiments, we investigated
how neurons influence the in vitro growth of resident macrophages belonging to the microglial population (BMs) and obtained from developing CNS. Figure 5B shows that neurons also
increased proliferation of BMs cultured in the presence of CSF-1, and
that stimulation of BM proliferation was inhibited by anti-TGF-2
antibodies. Thus, both BMs and BMMs exhibited the same biological
response to anti-TGF- antibodies.
None of the anti-TGF- antibodies tested interfered with neuron
survival or with CSF-1-stimulated macrophage proliferation when BMs and
BMMs were cultured in the absence of neuronal cells. The specificity
and efficiency of the TGF- antibodies was checked by Western blot or
the Mink lung assay test using recombinant isoforms (data not
shown).
rhTGF- isoforms 1-3 increase BMM and BM growth in the presence
of CSF-1
The ability of TGF- to increase CSF-1-dependent
proliferation of BMMs and BMs was investigated directly by adding
recombinant 1-3 isoforms of TGF- to the medium of macrophage
cultures. Figure 6 shows that each of the three isoforms
displayed the biological effects characterizing neuron-derived factors
in cocultures. In the case of BMMs (Fig. 6A),
significant stimulations were observed from concentrations of 10 pg/ml
(rhTGF-1) or 100 pg/ml (rhTGF-2 and -3). For each of the three
isoforms, optimal stimulations reached levels similar to those observed
in cocultures with neurons. As for BMs (Fig. 6B),
concentrations required for significant stimulation were 10 pg/ml
(rhTGF-3) and 100 pg/ml (rhTGF-1 and -2). The magnitudes of
optimal stimulation were similar for the three isoforms but were lower
than those obtained with BMMs. In the absence of CSF-1, none of the
TGF- isoforms allowed macrophages to survive to 96 hr (number of BMs
or BMMs less than five for each determination in cultures supplemented
with TGF-1-3 at concentrations up to 1 ng/ml). Kinetic analyses
performed with BMM cultures failed to reveal any short-term improvement
in cell survival when TGF-2 was added to cultures free of CSF-1
(Fig. 2).
Fig. 6.
Dose-response effect of recombinant TGF-s on
BMM (A) and BM (B) growth.
Cells were cultured for 96 hr in CDM supplemented with 2% FCS and 10 ng/ml rhCSF-1. TGF- isoforms were added daily to culture at
indicated final concentrations for the last 72 hr. The number of
macrophages in each condition is given as percentage of mean control
value obtained from wells without TGF- (set as 100%). Data are
mean ± SEM of five determinations from one experiment representative of five (BMM) or three (BM) independent experiments. Actual control numbers of BMs and BMMs were 54 ± 6 and 222 ± 9, respectively. Significant increases in macrophage number compared with control values were determined by ANOVA followed by Dunnett's test. BMM cultures (A), p < 0.01 from 0.01 ng/ml for TGF-1 and from 0.1 ng/ml for TGF-2 and
-3. BM cultures (B), p < 0.05 from 0.1 ng/ml for TGF-1, and p < 0.01 from 0.1 ng/ml for TGF-2 and from 0.01 ng/ml for TGF-3.
[View Larger Version of this Image (18K GIF file)]
Synthesis of TGF- by neurons of the developing
cerebral cortex
The expression of TGF- genes in our neuronal and macrophage
cultures was directly investigated by reverse transcription-PCR and
Southern blot analyses. Transcripts of TGF-2 and TGF-3 but not
TGF-1 genes were detected in neuronal cultures with or without addition of 10 ng/ml rhCSF-1 and 2% FCS to the medium (Fig.
7, lanes 2, 3). In contrast, BMs expressed
high levels of TGF-1 but not TGF-2 or TGF-3 transcripts (Fig.
7, lane 5), a result expected from previous studies with
cultured mouse BMs (Constam et al., 1992). TGF-1 transcripts were
also detected in BMMs, although the level of expression was lower (Fig.
7, lane 4).
Fig. 7.
Reverse transcription-PCR and Southern blot
analysis of TGF- mRNA in neurons and macrophages. Total RNA was
extracted from 4-d-old cultures of BMs, BMMs, or neurons derived from
E17 cerebral cortex. Autoradiograms were obtained by Southern
hybridization of amplified fragments as described in Materials and
Methods. Lane 1, Controls (mouse brain for TGF-1, rat
epithelial liver cell line for TGF-2, and peripheral nerve ganglions
mRNA for TGF-3); lanes 2 and 3,
neurons cultured in CDM without (lane 2) or with
(lane 3) 10 ng/ml rhCSF-1 and 2% FCS added for the last
72 hr; lane 4, BMMs; lane 5, BMs.
Macrophages were cultured in CDM supplemented with 10 ng/ml rhCSF-1 and
2% FCS.
[View Larger Version of this Image (46K GIF file)]
Considering the role of TGF-2 in our cocultures, neuronal expression
of this cytokine was further studied at the cellular level by
immunocytochemical detection of the protein. We took advantage of
monospecific anti-TGF-2 antibodies against human TGF-2, which was
previously used to label this cytokine in various mammalian tissues
(Anderson et al., 1995; Stewart et al., 1995; Frank et al., 1996).
Double immunostaining using anti-MAP2 antibodies revealed that >80%
of neurons in cultures derived from E17 cerebral cortex displayed
intracellular TGF-2 immunoreactivities (Fig. 8G,H). Staining with anti-TGF-2 was
restricted to a perinuclear area, which is likely to be the Golgi
apparatus (Fig. 8G); such a pattern is reminiscent of that
previously reported for cultured peripheral nerves (Stewart et al.,
1995).
Fig. 8.
Expression of TGF-2 by neurons of the cerebral
cortex in vivo and in vitro. A,
B, In situ hybridization dark-field
(A) and bright-field (B)
views of two different sections through the frontal part of the rat
cerebral cortex at postnatal day 6. Cortical layers II-V are marked. The dark-field view shows accumulation
of TGF-2 mRNA in cortical layers II and IV. Scale bar, 100 µm.
C-F, Double immunofluorescent staining of P6 frontal
cortex. C, E, anti-TGF-2 staining (FITC); D,
F, same fields stained with anti-MAP2 (TRITC). Scale bars:
C, D, 100 µm; E, F, 25 µm.
Arrows in E and F mark a
cell body double-stained with anti-MAP2 and anti-TGF-2. G, H, Double staining of neuronal cultures (4 d in
vitro) derived from E17 cerebral cortex with anti-TGF-2 mAb
(FITC; G) and anti-MAP2 mAb (TRITC,
H), Scale bar, 20 µm.
[View Larger Version of this Image (137K GIF file)]
TGF- is generally secreted as a latent TGF- complex unable
to bind to TGF- receptors and requiring extracellular activation for
biological activity (Flaumenhaft et al., 1993). In our coculture model,
the presence of TGF-2 activity indicates that cultured neurons
produce active forms of TGF-2 or that activation of neuron-derived latent TGF-2 occurs in the presence of macrophages. Using an ELISA
specific for active human TGF-2, we failed to detect accumulation of
mature TGF- isoforms in the medium assayed at the end of the cocultures (threshold for detection, 2 pg/ml using rhTGF-2). Mature
TGF-2 was detectable by ELISA in the medium of a pure neuronal
culture when cell densities were higher than those routinely used to
perform cocultures. Consistent with reverse transcription-PCR results,
TGF-2 was not detected in BM or BMM cultures (data not shown).
However, latent TGF-2 was repeatedly detected in the media assayed
at the end of cocultures and control neuronal cultures, after transient
acidification of the media, at mean concentrations ranging between 5 and 10 pg/ml (means from three wells for each culture; SD, <1
pg/ml).
The synthesis of TGF-2 by neurons of the cerebral cortex was also
investigated in tissue sections from developing brain. We chose to
focus our observations on postnatal day 6, a developmental stage that
corresponds to both infiltration and active proliferation of
macrophages in this CNS region (Ling and Wong, 1993). In
situ hybridization revealed TGF-2 mRNA expression in frontal,
parietal, and cingular cortices (Fig. 8). Hybridization signals
obtained with a TGF-2 antisense riboprobe displayed a laminar
pattern corresponding mostly to cortical layers II and IV (Fig.
8A,B). Controls performed using the corresponding
sense probe provided a weak and uniformly distributed nonspecific
background signal (not shown). TGF-2 immunoreactivity was detected
in adjacent tissue sections across all cortical layers but was more
intense in layer II (Fig. 8C). Double immunostaining with
anti-MAP2 antibodies allowed unambiguous localization of TGF-2
immunoreactivity to perinuclear regions of neuronal cell bodies (Fig.
8E,F). Weak staining with anti-TGF-2 was
also observed in cells scattered in the corpus callosum. Control
experiments for immunocytochemistry including peptide adsorption of the
anti-TGF-2 antibodies were negative (not shown).
DISCUSSION
Infiltration of the CNS by bone marrow-derived phagocytes
and the proliferation of macrophages are seminal events of microglial growth and pathological reactions (Hickey and Kimura 1988; Perry et
al., 1994). This recruitment of macrophages is supported by CSF-1, a
growth factor that is produced by astrocytes (Giulian and Ingeman,
1988; Hao et al., 1990; Théry et al., 1990; Roth and Stanley,
1992, 1996; Raivich et al., 1994).
The present study describes a novel biological mechanism by which
neurons regulate macrophage proliferation within the CNS. Using a
coculture assay, we show that neurons release compounds that increase
mitogenic responses of BMs and BMMs to CSF-1. This capacity is shared
by rat and mouse neurons of different brain regions such as the
cerebral cortex, cerebellum, and mesencephalon.
TGF-1-3 are highly conserved 25 kDa homodimers encoded by
distinct genes that present substantial amino acid sequence homologies (70-80%). These peptides modulate both functional properties and growth of mesenchymal and neuroepithelial cells (for review, see Sporn
and Roberts, 1992; Massagué et al., 1994; Krieglstein et al.,
1995a). Focusing on neurons of the cerebral cortex, TGF-2 produced
by these cells seems to be the primary, if not the unique, molecular
effector of macrophage proliferation. Indeed, the effect of neurons was
completely blocked in the presence of antibodies neutralizing TGF-2.
Purified TGF-2, added to BM and BMM cultures, was found to enhance
the mitogenic activity of CSF-1, and the secretion of TGF-2 by
cerebrocortical neurons was demonstrated by detection of the
transcripts and the protein. Although we did not attempt to confirm the
role of TGF-2 in coculture performed with neurons of cerebellum or
mesencephalon, our analysis is consistent with detection of TGF-2
gene expression in neuronal cultures derived from these two CNS regions
(Krieglstein et al., 1995b; De Luca et al., 1996).
TGF- is generally secreted as an inactive form resulting primarily
from its noncovalent association to the N-terminal cleavage product of
the TGF- precursor. Latent TGF- complex can be activated by
different means, including acid treatment and exposure to glycosidases and proteases (Flaumenhaft et al., 1993). ELISA determination confirmed
the production of latent TGF-2 by neurons from the cerebral cortex,
but this method did not allow detection of the mature form in our
coculture conditions. The quantitation of TGF-2 in our cultures
using an ELISA optimized for detection of human TGF-2 might suffer
from a reduced cross-reactivity with the rat-derived isoform. However,
our results support evidence indicating that latent TGF- can be
activated at the surface of different target cells by mechanisms that
can prevent the release of mature TGF- in a form detectable in a
conventional bioassay (Lucas et al., 1990; Saad et al., 1991; Arrick et
al., 1992; Flaumenhaft et al., 1993). In our experiments, generation of
mature TGF-2 at the macrophage surface could involve proteases or
extracellular matrix proteins such as plasmin and thrombospondin-1. In
fact, these compounds are known to activate TGF-, and they can be
produced by macrophages, including BMs (Nakajima et al., 1992a,b;
Flaumenhaft et al., 1993; Schultz-Cherry et al.,1993; Chamak et al.,
1994).
Previous studies focusing on TGF-1 have shown that this isoform
increases or reduces the mitogenic effect of CSF-1 depending on the
species, the tissue of origin, and the stage of differentiation of
cells from the macrophage lineage (Ohta et al., 1987; Celada and Maki,
1992; Suzumura et al., 1993; Rosenfeld, 1994). Here we found that
rhTGF-1-3 on their own do not support survival of rat BMs or
BMMs but markedly enhance the CSF-1-induced proliferation of these
phagocytes. This result is in line with TGF-1 stimulations reported
for adhering mouse BMMs (Celada and Maki, 1992), but our data are in
contrast with those of Suzumura et al. (1993), suggesting that TGF-1
reduces mitogenic effects of CSF-1 on mouse BMs. The common effect of
the various TGF- isoforms in our model is in agreement with the fact
that the various isoforms share promiscuous receptors displaying serine
threonine kinase activity (Massagué et al., 1994). TGF- is
known to be a potent chemoattractant acting on BMs and blood monocytes
(Wahl et al., 1987; Yao et al., 1990). In addition to cell attraction,
our results suggest that this cytokine also favors local expansion of
macrophages in the CNS by enhancing the effect of CSF-1. The molecular
mechanism of this synergy remains to be investigated. TGF- could
modulate the macrophage expression of the CSF-1 receptor and/or act
downstream at the level of intracellular transduction processes.
We have also observed that cultured neurons from the cerebral cortex
express TGF-2 and TGF-3 but not TGF-1 transcripts. However,
although rhTGF-3 is at least as potent as rhTGF-2 in promoting
macrophage proliferation in the presence of CSF-1, we found no evidence
that TGF-3 is involved in the neuronal effect. Indeed, the influence
of neurons on macrophages was abrogated by antibodies neutralizing
TGF-2 but not TGF-3. This indicates that the neuron-derived
TGF-3 did not reach sufficient concentrations in the cocultures to
stimulate macrophage growth. Any marginal contribution of this isoform
was further ruled out by the fact that monospecific antibodies blocking
TGF-3 did not modify the neuron effect on macrophage proliferation.
Previous studies have also failed to detect any production of TGF-3
in cultures of mouse astrocytes or cerebellar granule cells, despite
the expression of a TGF-3 transcript in these cultures (Constam et
al., 1992, 1994). In line with these observations, a
post-transcriptional downregulation of TGF-3 synthesis was
documented by identification of a 5 noncoding region of the TGF-3
mRNA which exerts a potent inhibitory effect on translational
efficiency in cell lines (Arrick et al., 1991).
The production of TGF- isoforms in the developing CNS has been
investigated in vivo (for review, see Krieglstein et al., 1995a). Except for the mouse cerebellum, in which expression of TGF-2 mRNA was studied during postnatal periods of development (Constam et al., 1994), the expression of TGF- genes in the
developing mammalian CNS has been studied mostly during prenatal stages
(Gatherer et al., 1990; Flanders et al., 1991; Millan et al., 1991;
Pelton et al., 1991; Schmid et al., 1991). TGF-2 and -3 mRNA were
also observed in the cerebral cortex of 1-d-old rats (Poulsen et al., 1994). Our own in situ detection of TGF-2 extends these
previous analyses to a postnatal stage when both the infiltration of
bone marrow-derived monocytes and intracerebral proliferation of
macrophages account for a marked expansion of microglia in the cerebral
cortex (Ling and Wong, 1993). In addition, we have demonstrated the
localization of TGF-2 in neuronal cell bodies by double staining
with antibodies raised against a neuronal marker. The in
situ detection of transcripts confirms the expression of the
TGF-2 gene in this developing region. High levels of both
transcripts and proteins were localized to layer II. However, the
protein appeared homogeneously distributed in the cortical layers
underneath, whereas TGF-2 transcript accumulated in layer IV (Fig.
8). Such a discrepancy in the amount of TGF-2 transcript and
translation products has previously been emphasized when proteins and
transcripts were compared in different regions of the peripheral
nervous system and the CNS (Pelton et al., 1991; Unsicker et al., 1991;
Stewart et al., 1995). The apparent lack of TGF-2 transcripts in
cells immunoreactive for TGF-2 could reflect technical limits for
the detection of low levels of mRNA using in situ
hybridization. Immunoreactivity of some cells could also stem from the
uptake of TGF-2 secreted in their vicinity.
Considering the expression of CSF-1 in the developing CNS (Théry
et al., 1990; Chang et al., 1994; Roth and Stanley, 1996), the present
study strongly suggests that neurons support microglial growth during
development by secreting TGF-2, which stimulates the proliferation
of BMs and their precursors infiltrating the CNS tissue. Furthermore,
our results provide a new functional significance for the intracerebral
synthesis of TGF-2. Beyond developmental topics, TGF-2 and CSF-1
expression has been detected in adult CNS, and studies performed with
CSF-1-deficient mice indicate that CSF-1 is required for the occurrence
of macrophages in the injured brain (Hulkower et al., 1991; Unsicker et
al., 1991; Raivich et al., 1994; Berezovskaya et al., 1995). Thus, the
synergic effect of CSF-1 and TGF- cytokines could contribute to the
macrophage reaction, which has been demonstrated in both experimental
lesions and a variety of human pathologies, including acquired
immunodeficiency syndrome and neurodegenerative diseases (Dickson et
al., 1993; McGeer et al., 1993; Perry et al., 1994). Noteworthy,
different types of CNS injuries are associated with an induction of
TGF-1 synthesis localized to reactive astroglial cells and
macrophages (Krieglstein et al., 1995a). Although the neuronal
expression of TGF-1 transcripts has also been observed as a
consequence of ischemia or cranial nerve transection (Lefebvre et al.,
1992; Knuckey et al., 1996), modulation of other neuronal TGF-
isoforms remains little studied. In this respect, the regulation of
neuronal secretion of TGF-2 and its biological effect under pathological conditions deserves further investigation.
FOOTNOTES
Received Jan. 27, 1997; revised April 24, 1997; accepted May 5, 1997.
This work was supported by Institut National de la Santé et de la
Recherche Médicale and a grant from Agence Nationale de Recherche
sur le SIDA. We thank Dr. Annette Koulakoff for participation in this work, Dr. Seillan-Heberden for providing the liver epithelial cell line, and Dr. Charles Félix Calvo for critical reading of this manuscript.
Correspondence should be addressed to Dr. Michel Mallat, Institut
National de la Santé et de la Recherche Médicale U 114, Chaire de Neuropharmacologie, Collège de France, 11 Place
Marcelin Berthelot, 75231 Paris Cedex 05, France.
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