Vol. 169, No. 2, 1990 June 15, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 719-724
PRODUCTION OF GRANULOCYTE/MACROPHAGECOLONY-STIMULATING
FACTOR
BY CULTURED ASTROCYTES
Kazushige
Ohno1r3, Akio
Suzumura'.
Makoto
Sawadal,
and Tohru Marunouchil
'Division
of Cell
Science,
Biology,
Institute
and'Department
Fujita 3Department
Health
Comprehensive
of Neurology,
University,
of Biological
Sciences,
for
Toyoake,
Chemistry
Nagoya City
School
of Medicine,
Aichi,
, Faculty
University,
Medical
Japan
of Pharmaceutical
Nagoya,
Japan
Received May 14, 1990
Summary: We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide(LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GMCSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF. 01990 Academic Press,Inc.
Granulocyte/macrophage interleukin-3 regulates
(IL-3) survival,
progenitor 3-like
cells factor
controlling
the
IL-3-like
to
growth
and differentiation
In the
reportedly astrocytes
migratory
phagocytes
(2,3). factor
factor
belong
(1). is
(LPS)-stimulated
brain
colony-stimulating a family
of glycoprotein
central
in
and may play
However, produced
of
nervous
produced
and proliferative
haemopoietic (CNS),
IL-
lipopolysaccharide an important
was still
by astrocytes
and
CSF that
system
functions it
(GM-CSF)
of
role
mononuclear
unclear is
IL-3
in
whether itself
or
0006-291X/90$1.50 719
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
169,
No.
another to
2, 1990
factor
identify
astrocytes for
the
the
IL-3-
BIOCHEMICAL
with the
did
AND
IL-3-like factor
not
activity.
and
produce
astrocyte-derived
BIOPHYSICAL
found
IL-3
but
IL-3-like
and GM-CSF-dependent
MATERIALS
cell
RESEARCH
In that that
study
we tried
LPS-stimulated GM-CSF
activity line
this
COMMUNICATIONS
of
was
murine responsible
fostering
IC-2,
(4).
AND METHODS
Cell cultures : Astrocytes were prepared from the primary mixed glial cell cultures of newborn C3H/HeN mice,as Briefly, after removal of the meninges, the described (5). brains were mechanically dissociated by nylon mesh. The cells were seeded in culture medium (Eagle's MEM suplemented with 10% fetal calf serum, 0.5 pg/ml of bovine insulin and 0.2% glucose) in 75-cm2 tissue culture flasks at the concentration of 2 brains per flask. After 14 days in culture, astrocytes were purified by 3-4 times repetitions of trypsinization and replating of the primary glial cell cultures. Purity of astrocytes was more than 95% as determined by indirect immunofluorescence assay with an a monoclonal antibody against glial fibrillary astrocyte marker, acidic protein (GFAP). Production of IL-3-like factor by astrocytes : Isolated astrocytes were seeded in 24-well test plates at a density of 1 x lo5 cells/500 ~1 in each well. After 12h in culture they were stimulated with various concentrations of lipopolysaccharide (from E.coli, DIFCO LABORATORIES, Detroit, USA). IC-2 cells (1 x 104/100 ~1 in each well) were cultured in triplicate in 96-well test plates together with either 50 ~1 of test samples or graded doses of recombinant IL-3 or GM-CSF. In the neutralization experiments, anti-mouse IL-3 monoclonal antibody (generously provided by Dr. John Abrams, DNAX Research Inst. of Molecular and Cellular Biology, Palo Alto, CA) (6) and/or anti-mouse GM-CSF antiserum (provided by Dr.J. Schreurs, DNAX, Palo Alto, CA) was added at 1:200 or 1:lOOO dilution as the final respective concentration. These antibodies specifically recognized the antigen and did not show crossreactivity After 48h incubation at 37OC, the (7). proliferation of IC-2 cells was assessed by MTT calorimetric assay as detailed previously (8). MTT is cleaved by living cells to yield a formazan product. Thus the measurement of MTT formazan products at OD 620nm by an Immuno Reader (Inter Med Japan, Tokyo, Japan) represents the number of live cells. Northern blot analysis : Expression of IL-3 and GM-CSF mRNA was assessed by Northern blot analysis. Total cellular RNA was isolated by lysing cells in guanidium isothiocyanate and RNA was recovered by centrifugation through cesium chloride (9) . Then 20 pg / sample were fractionated into 1.5% agarose gel with 6% formaldehyde and blotted onto nylon membrane (Hybond-N, Amersham Japan, Tokyo, Japan). RNA from EL-4 cells (5 x 105 /ml) stimulated with TPA (0.1 pg/ml) for 4 h and RNA from WEHIwas used as positive control for GM-CSF and IL-3 mRNA expression, respectively. After 100 pg/ml salmon sperm DNA and 1 x 106 cpm/ml of labeled probe had been added to the solution, hybridization was performed at 42'C in a solution containing 0.1 M sodium-PIPES, 0.1% SDS, 5 mM EDTA, 5 x Denhardt's solution, 720
Vol.
169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
10% dextran sulfate and 50% deionized formamide. The membranes were then washed twice for 30 min in 2 x SSC/l% SDS at 65'C and finally exposed to film with an intensifying screen at -8O'C for 24h. The Xho 1 - Xho 1 fragments of murine GM-CSF (10) and IL-3 cDNA (11.) obtained from DNAX Research Institute of Molecular and Cellular Biology (Palo Alto, Ca, U.S.A.) were labeled for a specific activity of log cpm/pg by using hexanucleotide primer and CX[~~P]-~CTP.
RESULTS IL-3-like
activity
stimulated at
astrocytes
a concentration
LPS at
we employed
In
from
the
stimulation
1 pg/ml.
was cytotoxic
to
with
supernatant with
Since
astrocytes
10 kg/ml
of
LPS-
LPS for
stimulation
24h with
(data
not
shown),
for
24h
in
LPS
the
experiments.
corder to
IC-2
in
1) by
more than
stimulation
monoclonal the
(Fig. of
100 pg/ml
subsequent
was detected
identify
antibody
or
proliferation
LPS-stimulated
Anti-GM-CSF
the
IL-3-like
anti-mouse assay
or
completely
anti-mouse
GM-CSF antiserum
system
astrocytes
antiserum
factor,
with
culture
non-stimulated blocked
the
IL-3
was added to supernatants astrocytes.
activity
of
the
Anti-IL-3 Anti-GM-CSF
01
lo-’16’lo-’10” lo1lrf (lg/ml)
Anti-GM-CSF L anti-IL-3
' 0.00
02 factor
0.02
0.04
0.06
0.08
OD620nm
Kinetics of IL-3-like induction by LPS. Astrocytes were stimulated with various concentrations of LPS for 24h, 48h and 72h. Supernatant IL-3-like activity was assayed Each point represents as described in MATERIALS AND METHODS. the mean of 3 assays (& SD). Fiq. 2. Inhibition of IL-3-like activity by specific anti.bodies. Anti-IL-3 antibody (1:200) or anti-GM-CSF antibody was added to the supernatant from unstimulated (1:1000) Their astrocytes or LPS (10 pg/ml)-stimulated astrocytes. effect on the proliferation of IC-2 cells was assayed by MTT Each bar represents the mean of 3 assays (+calorimetric assay. SD).
Fiq.
1.
721
1
O.iO
Vol.
169, No. 2, 1990
factor
produced
anti-IL-3 (Fig.
BIOCHEMICAL
by LPS-stimulated
monoclonal 21,
cell
growth
that
the
IL-3
itself.
this
of
IC-2
astrocytes
IL-3
(data
not
(Fig. 16 h,
mRNA was
stimulation
with
of
not
block
LPS (Fig.
activity
at
dependent
These
results
suggest
GM-CSF,
and not
factor
in
is
examined
detected
by Northern
in
stimulation
untreated
with
10 pg/ml
GM-CSF mRNA (Fig.
astrocytes,
not
even
3). after
3).
DISCUSSION GM-CSF is cells,
fibroblasts
a cytokine or
from
activated
macrophages,
T cells,
and controls
endothelial proliferation
6
A
28s 28s-t 18s 18s-9
0 16
all
shown).
expressed
detected
However,
IL-3
after
astrocytes
the
2).
blocked
mRNA was not
However,
(Fig.
completely
GM-CSF gene was then
3). the
not
IL-3-like
GM-CSF
RESEARCH COMMUNICATIONS
astrocytes
did
antibody
astrocyte-derived
analysis.
LPS for
antibody
while
The expression blot
AND BIOPHYSICAL
C
c
Fiq. 3. astrocytes.
0 4
8 16
Expression of GM-CSF gene (A) and IL-3 gene (B) in (A) Total cellular RNA from unstimulated (column 0) and LPS-stimulated cells was collected 16h after induction (column 16) and analyzed by Northern blot (20 pg per lane). Column c: total cellular RNA from EL-4 cells stimulated with TPA for 4h. The position of GM-CSF mRNA is indicated by a thick arrow. (B) Total cellular RNA from unstimulated (column 0) and LPS-stimulated cells was isolated at 4, 0 and 16h (column 4, 8, 16) and analyzed by Northern blot (20 pg per lane). Column c: total cellular RNA from WEHIcells. The position of IL-3 mRNA is indicated by a thick arrow. 722
BIOCHEMICAL
Vol. 169, No. 2, 1990
and differentiation suggested
of
to
infection
play
(1).
response
to
(5)
is
that
by astrocytes.
in
the
This
CSF (12,13),
cultured in
situ
regions
that
it
possible
et
al.
AL-3
in
detect Neither system, factor
that
microglia
detected
astrocytes
in
cells
to
microglia
(brain
observation),
production
that
purify
it
of
GM-CSF
GM-CSF production role
However,
of
by
GM-CSF
since
we and
proliferate
in response
to GM-
GM-CSF from
astrocytes
in
the
in
normal
induces
developmental
the IL-3
that
mouse brain, Neurons
IL-3
or other
cultured
rat
astrocytes
and
and performed
expression
of
murine
mRNA was detected
that
GM-CSF, and not
astrocytes
which
of
the
regulates
wide
neuroncells
this
blot
purified
IL-3,
in
may
mRNA was not
IL-3
in
of
shown by
expressed
In
Northern
IL-3
have
IL-3
for
mRNA in
al.
of glial
(15).
specific
a supernatant
cells
However,
supernatant
IL-3
in
populations
vivo.
antibody
et
mRNA is
especially
mRNA in
nor
in
Farrar
IL-3
indicating from
activity
Recently
(14)
a monoclonal the
IL-3-like
(2).
hybridization
express
emptiyed
GM-CSF in vitro
conditions.
of
detected
of
The precise
microglia
of
dense regions. thus
time
elucidation.
found
proliferation
Frei
first
in
and
induction
adherent
a role
is the
further
is
the
LPS-stimulated
has been demonstrated.
groups
diseased
produce
play
also
inflammation
GM-CSF (unpublished
microglia
(CNS awaits
other
the
produce
in
GM-CSF is
through
deleted
and we have found
do not
astrocytes
study
mouse astrocytes we have
(1).
role
our
RESEARCH COMMUNICATIONS
cells
important
Since
macrophage) unlikely
an
that
LPS.
astrocytes
haemopoietic
We showed in
gene expression
AND BIOPHYSICAL
our
study,
to
we
neutralize analysis
to
astrocytes. experimental
is most likely microglia
the in
the
brain.
ACKNOWLEDGMENTS We are grateful to Drs. Jolanda Schreurs and John Abrams, DNAX Research Inst. of Molecular and Cellular Biology, CA, U.S.A., for providing us with anti-GM-CSF and anti-IL-3 for the permission to use antibodies. We also thank DNAX Inst. murine IL-3 cDNA and GM-CSF cDNA. 723
Vol. 169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
This study was supported in part by Grants-in-Aid Japanese Ministry of Education, the Human Science Foundation and the Fujita Health University.
from the Research
REFERENCES 1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15.
Nicola,N.A. (1989) Annu. Rev. Biochem., 58, 45-77. Frei,K., Bodmer, S., Schwerdel,C. and Fontana,A. (1985) J.Immunol., 135, 4044-4041. Frei,K., Bodmer, S., Schwerdel,C. and Fontana,A. (1986) J.Immunol., 137, 3521-3527. Koyasu, S., Miyajima, A., Arai,K., Okajima,M.U. and Yahara, I. (1989) Cell struct.Funct., 14, 459-471. Sawada,M., Kondo,N., Suzumura,A. and Marunouchi,T. (1989) Brain Res., 491, 394-397. Abrams,J.S., and Pearce,M.K. (1988) J.Immunol., 140, 131135. Quill,H., Gaur,A. and Phipps,R.P. (1989) J.Immunol., 142, 813-818. Sawada,M., Suzumura,A., and Marunouchi,T. Yamamoto,H. (1990) Brain Res, 509, 119-124. Chirgwin,J., Pryzbyla,A., Mcdonald,R. and Rutter,W. (1979) Biochemistry, 18, 5294-5299. Yokota,T., Lee,F., Rennick,D., Hall,C., Arai,N., Mosmann, T ., Nabel,G., Canter,H. and Arai,K. (1984) Proc.Natl.Acd. Sci.USA, 81, 1070-1074. Miyatake,S., Otsuka,T., Lee,F. and Arai,K. (1985) EMBO J., 4, 2561-2568. Giulian,D. (1988) J.Neurosci., 8, 4707and Ingeman, J.E. 4717. Suzumura,A., Sawada,M., Yamamoto,H. and Marunouchi,T., submitted for publication. Farrar,W.L., Vinocour,M. and Hil1,J.M. (1989) Blood, 73, 137-140. Lieberman,A., Pitha,P.M., Shin,H. and Shin,M. (1989) Proc. Natl.Acad.Sci.USA, 86, 6348-6352.
724
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 719-724
PRODUCTION OF GRANULOCYTE/MACROPHAGECOLONY-STIMULATING
FACTOR
BY CULTURED ASTROCYTES
Kazushige
Ohno1r3, Akio
Suzumura'.
Makoto
Sawadal,
and Tohru Marunouchil
'Division
of Cell
Science,
Biology,
Institute
and'Department
Fujita 3Department
Health
Comprehensive
of Neurology,
University,
of Biological
Sciences,
for
Toyoake,
Chemistry
Nagoya City
School
of Medicine,
Aichi,
, Faculty
University,
Medical
Japan
of Pharmaceutical
Nagoya,
Japan
Received May 14, 1990
Summary: We investigated the production of interleukin-3 (IL-3)-like factor by murine astrocytes. Supernatants from lipopolysaccharide(LPS)-stimulated astrocytes induced proliferation of IC-2, an IL-3and granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent cell line. This activity was completely neutralized by the antibody against GMCSF but not by the anti-IL-3 monoclonal antibody. Northern blot analysis revealed the expression of GM-CSF mRNA, but not of IL-3 mRNA, in cultured astrocytes. These results indicate that with proper stimuli murine astrocytes produce GM-CSF. 01990 Academic Press,Inc.
Granulocyte/macrophage interleukin-3 regulates
(IL-3) survival,
progenitor 3-like
cells factor
controlling
the
IL-3-like
to
growth
and differentiation
In the
reportedly astrocytes
migratory
phagocytes
(2,3). factor
factor
belong
(1). is
(LPS)-stimulated
brain
colony-stimulating a family
of glycoprotein
central
in
and may play
However, produced
of
nervous
produced
and proliferative
haemopoietic (CNS),
IL-
lipopolysaccharide an important
was still
by astrocytes
and
CSF that
system
functions it
(GM-CSF)
of
role
mononuclear
unclear is
IL-3
in
whether itself
or
0006-291X/90$1.50 719
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
169,
No.
another to
2, 1990
factor
identify
astrocytes for
the
the
IL-3-
BIOCHEMICAL
with the
did
AND
IL-3-like factor
not
activity.
and
produce
astrocyte-derived
BIOPHYSICAL
found
IL-3
but
IL-3-like
and GM-CSF-dependent
MATERIALS
cell
RESEARCH
In that that
study
we tried
LPS-stimulated GM-CSF
activity line
this
COMMUNICATIONS
of
was
murine responsible
fostering
IC-2,
(4).
AND METHODS
Cell cultures : Astrocytes were prepared from the primary mixed glial cell cultures of newborn C3H/HeN mice,as Briefly, after removal of the meninges, the described (5). brains were mechanically dissociated by nylon mesh. The cells were seeded in culture medium (Eagle's MEM suplemented with 10% fetal calf serum, 0.5 pg/ml of bovine insulin and 0.2% glucose) in 75-cm2 tissue culture flasks at the concentration of 2 brains per flask. After 14 days in culture, astrocytes were purified by 3-4 times repetitions of trypsinization and replating of the primary glial cell cultures. Purity of astrocytes was more than 95% as determined by indirect immunofluorescence assay with an a monoclonal antibody against glial fibrillary astrocyte marker, acidic protein (GFAP). Production of IL-3-like factor by astrocytes : Isolated astrocytes were seeded in 24-well test plates at a density of 1 x lo5 cells/500 ~1 in each well. After 12h in culture they were stimulated with various concentrations of lipopolysaccharide (from E.coli, DIFCO LABORATORIES, Detroit, USA). IC-2 cells (1 x 104/100 ~1 in each well) were cultured in triplicate in 96-well test plates together with either 50 ~1 of test samples or graded doses of recombinant IL-3 or GM-CSF. In the neutralization experiments, anti-mouse IL-3 monoclonal antibody (generously provided by Dr. John Abrams, DNAX Research Inst. of Molecular and Cellular Biology, Palo Alto, CA) (6) and/or anti-mouse GM-CSF antiserum (provided by Dr.J. Schreurs, DNAX, Palo Alto, CA) was added at 1:200 or 1:lOOO dilution as the final respective concentration. These antibodies specifically recognized the antigen and did not show crossreactivity After 48h incubation at 37OC, the (7). proliferation of IC-2 cells was assessed by MTT calorimetric assay as detailed previously (8). MTT is cleaved by living cells to yield a formazan product. Thus the measurement of MTT formazan products at OD 620nm by an Immuno Reader (Inter Med Japan, Tokyo, Japan) represents the number of live cells. Northern blot analysis : Expression of IL-3 and GM-CSF mRNA was assessed by Northern blot analysis. Total cellular RNA was isolated by lysing cells in guanidium isothiocyanate and RNA was recovered by centrifugation through cesium chloride (9) . Then 20 pg / sample were fractionated into 1.5% agarose gel with 6% formaldehyde and blotted onto nylon membrane (Hybond-N, Amersham Japan, Tokyo, Japan). RNA from EL-4 cells (5 x 105 /ml) stimulated with TPA (0.1 pg/ml) for 4 h and RNA from WEHIwas used as positive control for GM-CSF and IL-3 mRNA expression, respectively. After 100 pg/ml salmon sperm DNA and 1 x 106 cpm/ml of labeled probe had been added to the solution, hybridization was performed at 42'C in a solution containing 0.1 M sodium-PIPES, 0.1% SDS, 5 mM EDTA, 5 x Denhardt's solution, 720
Vol.
169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
10% dextran sulfate and 50% deionized formamide. The membranes were then washed twice for 30 min in 2 x SSC/l% SDS at 65'C and finally exposed to film with an intensifying screen at -8O'C for 24h. The Xho 1 - Xho 1 fragments of murine GM-CSF (10) and IL-3 cDNA (11.) obtained from DNAX Research Institute of Molecular and Cellular Biology (Palo Alto, Ca, U.S.A.) were labeled for a specific activity of log cpm/pg by using hexanucleotide primer and CX[~~P]-~CTP.
RESULTS IL-3-like
activity
stimulated at
astrocytes
a concentration
LPS at
we employed
In
from
the
stimulation
1 pg/ml.
was cytotoxic
to
with
supernatant with
Since
astrocytes
10 kg/ml
of
LPS-
LPS for
stimulation
24h with
(data
not
shown),
for
24h
in
LPS
the
experiments.
corder to
IC-2
in
1) by
more than
stimulation
monoclonal the
(Fig. of
100 pg/ml
subsequent
was detected
identify
antibody
or
proliferation
LPS-stimulated
Anti-GM-CSF
the
IL-3-like
anti-mouse assay
or
completely
anti-mouse
GM-CSF antiserum
system
astrocytes
antiserum
factor,
with
culture
non-stimulated blocked
the
IL-3
was added to supernatants astrocytes.
activity
of
the
Anti-IL-3 Anti-GM-CSF
01
lo-’16’lo-’10” lo1lrf (lg/ml)
Anti-GM-CSF L anti-IL-3
' 0.00
02 factor
0.02
0.04
0.06
0.08
OD620nm
Kinetics of IL-3-like induction by LPS. Astrocytes were stimulated with various concentrations of LPS for 24h, 48h and 72h. Supernatant IL-3-like activity was assayed Each point represents as described in MATERIALS AND METHODS. the mean of 3 assays (& SD). Fiq. 2. Inhibition of IL-3-like activity by specific anti.bodies. Anti-IL-3 antibody (1:200) or anti-GM-CSF antibody was added to the supernatant from unstimulated (1:1000) Their astrocytes or LPS (10 pg/ml)-stimulated astrocytes. effect on the proliferation of IC-2 cells was assayed by MTT Each bar represents the mean of 3 assays (+calorimetric assay. SD).
Fiq.
1.
721
1
O.iO
Vol.
169, No. 2, 1990
factor
produced
anti-IL-3 (Fig.
BIOCHEMICAL
by LPS-stimulated
monoclonal 21,
cell
growth
that
the
IL-3
itself.
this
of
IC-2
astrocytes
IL-3
(data
not
(Fig. 16 h,
mRNA was
stimulation
with
of
not
block
LPS (Fig.
activity
at
dependent
These
results
suggest
GM-CSF,
and not
factor
in
is
examined
detected
by Northern
in
stimulation
untreated
with
10 pg/ml
GM-CSF mRNA (Fig.
astrocytes,
not
even
3). after
3).
DISCUSSION GM-CSF is cells,
fibroblasts
a cytokine or
from
activated
macrophages,
T cells,
and controls
endothelial proliferation
6
A
28s 28s-t 18s 18s-9
0 16
all
shown).
expressed
detected
However,
IL-3
after
astrocytes
the
2).
blocked
mRNA was not
However,
(Fig.
completely
GM-CSF gene was then
3). the
not
IL-3-like
GM-CSF
RESEARCH COMMUNICATIONS
astrocytes
did
antibody
astrocyte-derived
analysis.
LPS for
antibody
while
The expression blot
AND BIOPHYSICAL
C
c
Fiq. 3. astrocytes.
0 4
8 16
Expression of GM-CSF gene (A) and IL-3 gene (B) in (A) Total cellular RNA from unstimulated (column 0) and LPS-stimulated cells was collected 16h after induction (column 16) and analyzed by Northern blot (20 pg per lane). Column c: total cellular RNA from EL-4 cells stimulated with TPA for 4h. The position of GM-CSF mRNA is indicated by a thick arrow. (B) Total cellular RNA from unstimulated (column 0) and LPS-stimulated cells was isolated at 4, 0 and 16h (column 4, 8, 16) and analyzed by Northern blot (20 pg per lane). Column c: total cellular RNA from WEHIcells. The position of IL-3 mRNA is indicated by a thick arrow. 722
BIOCHEMICAL
Vol. 169, No. 2, 1990
and differentiation suggested
of
to
infection
play
(1).
response
to
(5)
is
that
by astrocytes.
in
the
This
CSF (12,13),
cultured in
situ
regions
that
it
possible
et
al.
AL-3
in
detect Neither system, factor
that
microglia
detected
astrocytes
in
cells
to
microglia
(brain
observation),
production
that
purify
it
of
GM-CSF
GM-CSF production role
However,
of
by
GM-CSF
since
we and
proliferate
in response
to GM-
GM-CSF from
astrocytes
in
the
in
normal
induces
developmental
the IL-3
that
mouse brain, Neurons
IL-3
or other
cultured
rat
astrocytes
and
and performed
expression
of
murine
mRNA was detected
that
GM-CSF, and not
astrocytes
which
of
the
regulates
wide
neuroncells
this
blot
purified
IL-3,
in
may
mRNA was not
IL-3
in
of
shown by
expressed
In
Northern
IL-3
have
IL-3
for
mRNA in
al.
of glial
(15).
specific
a supernatant
cells
However,
supernatant
IL-3
in
populations
vivo.
antibody
et
mRNA is
especially
mRNA in
nor
in
Farrar
IL-3
indicating from
activity
Recently
(14)
a monoclonal the
IL-3-like
(2).
hybridization
express
emptiyed
GM-CSF in vitro
conditions.
of
detected
of
The precise
microglia
of
dense regions. thus
time
elucidation.
found
proliferation
Frei
first
in
and
induction
adherent
a role
is the
further
is
the
LPS-stimulated
has been demonstrated.
groups
diseased
produce
play
also
inflammation
GM-CSF (unpublished
microglia
(CNS awaits
other
the
produce
in
GM-CSF is
through
deleted
and we have found
do not
astrocytes
study
mouse astrocytes we have
(1).
role
our
RESEARCH COMMUNICATIONS
cells
important
Since
macrophage) unlikely
an
that
LPS.
astrocytes
haemopoietic
We showed in
gene expression
AND BIOPHYSICAL
our
study,
to
we
neutralize analysis
to
astrocytes. experimental
is most likely microglia
the in
the
brain.
ACKNOWLEDGMENTS We are grateful to Drs. Jolanda Schreurs and John Abrams, DNAX Research Inst. of Molecular and Cellular Biology, CA, U.S.A., for providing us with anti-GM-CSF and anti-IL-3 for the permission to use antibodies. We also thank DNAX Inst. murine IL-3 cDNA and GM-CSF cDNA. 723
Vol. 169, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
This study was supported in part by Grants-in-Aid Japanese Ministry of Education, the Human Science Foundation and the Fujita Health University.
from the Research
REFERENCES 1. 2. 3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15.
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