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1991 78: 103-109
Peptide growth factors stimulate macrophage colony-stimulating
factor in murine stromal cells
SL Abboud and M Pinzani
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Peptide Growth Factors Stimulate Macrophage Colony-Stimulating Factor in
Murine Stromal Cells
By Sherry L. Abboud and Massimo Pinzani
Bone marrow stromal cells influence hematopoiesis through
cell-cell interaction and release of hematopoietic growth
factors. Macrophage colony-stimulating factor (M-CSF) is
constitutively produced by several murine and human stromal cell lines and is induced by inflammatory mediators such
as interleukin-1 a or tumor necrosis factor-a (TNF-a) in a
variety of mesenchymal cells. Other potentially important
regulatory molecules such as platelet-derived growth factor
(PDGF) and basic fibroblast growth factor (bFGF), released
by activated monocytes in response to inflammation, stimulate the growth of human stromal cells. However, the effect
of these peptide mitogens on M-CSF expression in stromal
cells has not been explored. In this study, we used TC-1
murine bone marrow-derived stromal cells that constitutively secrete M-CSF t o determine the effect of PDGF and
bFGF on cell proliferation and M-CSF gene expression. PDGF
and bFGF, but not TNF-a. were potent mitogens for the TC-1
cells. Similar to mouse L cells, TC-1 murine stromal cells
constitutively expressed two major mRNA transcripts of 4.4
and 2.2 kb that hybridized t o a murine M-CSF cDNA. PDGF,
bFGF, and TNF-a markedly stimulated the steady-state expression of M-CSF mRNA with different time-course kinetics.
The increased expression of M-CSF mRNA was associated
with enhanced secretion of M-CSFas determined by radioimmunoassay. These findings suggest that PDGF, bFGF, and
TNF-a may regulate hematopoiesis indirectly through release
of M-CSF by stromal cells and may modulate, at least in part,
the hematopoietic response t o inflammation.
0 1991by The American Society of Hematology.
H
vein endothelial cells. Others have shown that TNF as well
as GM-CSF, IL-3, and y-interferon induce M-CSF transcripts and M-CSF secretion in purified peripheral blood
In vivo, TNF stimulates the production of
M-CSF mRNA in certain tissues and raises the serum level
of M-CSF.”
The peptide growth factors, platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF), are
prime candidates that may affect the production of hematopoietic growth factors such as M-CSF. Both have a
broad specificity for a number of cells, including fibroblasts,
microvascular endothelial, and smooth muscle cells, resulting in their growth and
They are secreted
by activated monocytes or macrophages and have been
shown to induce the synthesis and release of other cytokines in target
To study M-CSF production by
stromal cells we have used, as a model, the TC-1 murine
bone marrow-derived stromal cells that constitutively secrete M-CSF.9331
We demonstrate that PDGF and bFGF
stimulate the induction of M-CSF mRNA levels and the
release of M-CSF in TC-1 cells. They also are potent
mitogens for these cells.
EMATOPOIETIC PROGENITORS proliferate and
differentiate in response to well-defined hematopoietic growth factors such as macrophage colony-stimulating
factor (M-CSF),’ granulocyte-macrophage CSF (GMCSF), granulocyte CSF (G-CSF), and interleukin-3 (IL3).’,’ Bone marrow stromal cells play a crucial role in
regulating hematopoiesis through direct cell-cell interacThe
tion and the release of hematopoietic growth factors.334
murine stroma is an integrated heterogeneous cell population composed primarily of fibroblasts, endothelial cells,
and macrophages.’ Studies performed on cell types thought
to be representative of the hematopoietic stromal microenvironment, including human dermal or embryonic lung
fibroblasts, human umbilical vein endothelial cells, and
peripheral blood monocytes, have shown that they constitutively secrete and/or express mRNAs that encode for CSFs,
including M-CSF.6The adherent cell layers of unstimulated
murine and human long-term bone marrow cultures have
also been shown to release colony-stimulating activity
(CSA).’ More recently, certain cloned murine stromal cell
lines have been reported to constitutively produce M-CSF
as well as other hematopoietic growth factors.’ lo M-CSF
was initially purified to homogeneity from L-cell-conditioned medium and shown to be a glycosylated disulfidelinked dimer of 70 Kd.” A cDNA encoding murine and
human M-CSF have been isolated and the recombinant
factors have been expressed in eukaryotic cell^.^'^'^ M-CSF
enhances mononuclear phagocyte survival, proliferation,
differentiation, and phagocytic and tumoricidal activities.I4-l6It also interacts synergistically with other growth
factors such as GM-CSF, IL-1 a,and IL-3 to stimulate early
murine hematopoietic colony-forming cells.”~”In contrast
to other CSFs, M-CSF is present in the circulation, suggesting that it may play an important role in regulating
hematopoiesis in ~ i v 0 . l ~
The production of M-CSF in response to several cytokines has been studied in certain cell types. However, the
regulation of M-CSF gene expression by biologic mediators
specifically in stromal cells has not been extensively examined. Seelentag et alZoidentified tumor necrosis factor
(TNF) and IL-1 as stimuli of M-CSF in human umbilical
Blood, Vol78, No 1 (July l), 1991:pp 103-109
~
From the Departments of Pathology and Medicine, VeteransAdministration Medical Center, Case Western Reserve University, Cleveland,
OH.
Submitted October 19, 1990; accepted March 6, 1991.
Supported by Public Health Service Grant P3OCA43703 awarded by
the National Cancer Institute and by the Veterans Administration
Research Service.
Portions of this work were presented in an abstmct form at the
annual meeting of the American Society of Hematology, Atlanta, GA,
December 2-5, 1989.
Address reprint requests to Sheny L. Abboud, MD, University of
Texas Health Science Center, Department of Medicine, 7703 Floyd
Curl Dr, San Antonio, TX 78284.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0OO6-497IJ91J7801-0002$3.
OOJO
103
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104
ABEOUD AND PINZANI
MATERIALS AND METHODS
Peptide growth factors and M-CSFprobe. Recombinant PDGF
(PDGF BB homodimer c-sis), recombinant human bFGF, and
TNF-a were purchased from Amgen Biologicals (Thousand Oaks,
CA). The specific activity of the TNF preparation was greater than
lo’ U/mg. Each unit represents the concentration of TNF required
to yield 50% lysis of mitomycin C-treated L929 mouse fibroblasts.
The M-CSF cDNA probe is a 2.4-kb fragment inserted into the
EcoRI site of SP65 and was a generous gift of Dr S. Clark (Genetics
Institute, Cambridge, MA).”
Stromal cell line. The TC-1 stromal cells (kindly provided by Dr
Peter Quesenberry, University of Virginia, Charlottesville) are
adherent cells isolated from murine long-term marrow culture.
Their phenotypic characterization has been previously described?
The cells were maintained in Fischer’s medium (GIBCO, Grand
Island, NY) supplemented with 10 mmol/L HEPES, 2 mmoVL
glutamine, 1 mmol/L sodium pyruvate, penicillin 100 U/mL,
streptomycin 100 kg/mL, nystatin 25 ng/mL, and 17% fetal calf
serum (FCS) (Hyclone, Logan, UT) and incubated at 37°C in 5%
CO,. Cells grown to confluency were passed weekly by exposure to
0.1% trypsin (GIBCO).
DNA synthesis. [3H]-thymidine (TdR) incorporation into the
TC-1 cells was used as a measure of DNA synthesis. In brief, 5 x
lo4 cells suspended in 1 mL of Fischer’s medium with 17% FCS
were seeded into each of 24-well flat-bottomed dishes and incubated at 37°C in a humidified atmosphere in 5% CO,. Confluent
cells were allowed to become quiescent by placing them in
Fischer’s medium with 1% serum for 48 hours. Cells were incubated with or without various growth factors for 20 hours and then
pulsed for 4 hours with 1.0 kCi/mL of [’HI-TdR (6.7 ci/mmol; New
England Nuclear, Boston, MA). In some experiments, as specified,
cells were pulsed with [’HI-TdR at the time growth factors were
added. The assay was terminated by gently aspirating the medium
and washing the cells three times with ice-cold 5% trichloroacetic
acid to precipitate proteins and nucleic acids and remove unincorporated [3H]-TdR. Cells were solubilized by adding 0.7 mL of 0.25
N NaOH in 0.1% sodium dodecyl sulfate (SDS). Aliquots of 0.5 mL
were then neutralized and isotope uptake was determined by liquid
scintillation counting.
Autoradiography. Stromal cells were plated onto 4-chamber
LabTek slides (Miles Scientific, Naperville, IL) at a density of 1 X
10‘ cells per chamber in Fischer’s medium supplemented with
serum. Confluent cells were made quiescent as described previously and then incubated with various growth factors and 1 pCi/mL
of [’HI-TdR for 24 hours. At the end of the pulsing period, an equal
volume of freshly prepared 3:l methano1:acetic acid fixative was
added to the medium for 10 minutes.” This half-strength fixative
was then replaced by an equal volume of undiluted 3:l methanol:
acetic acid fixative. After 10 minutes, cells were air dried and
exposed to NTB-2 nuclear emulsion (Kodak, Rochester, NY)for 3
days at 4°C. The slides were then developed and fixed with Kodak
D19 developer and Kodak fixer, respectively, and stained with
Giemsa.” Four hundred cells per each incubation condition were
counted and the percent of labeled nuclei (labeling index) was
determined.
Stromal cell proliferation. Stromal cells were seeded into 12well dishes at a density of 1 x 10s cellsiwell in Fischer’s medium
with 17% FCS. After 24 hours, media was aspirated and replaced
by Fischer’s medium containing 1% serum. At this time, test
conditions were added to each well (time 0). Cells in each well were
trypsinized and cell counts were performed on triplicate wells at
time 0 and after 3 and 7 days. Results were expressed as the
mean ? SE.
RNA purification and Northem analysis. Cells, 5 X lo5 to 1 X
lo6, suspended in complete medium were seeded into 100-mm
Petri dishes. At confluency, cells were made quiescent by incuba-
tion in 1% serum overnight. Cells were then incubated in the
absence or presence of growth factors. At specified time intervals,
stromal cells were washed twice in phosphate-buffered saline
(PBS), lysed with 5 m o w guanidium thiocyanate, and the RNA
recovered after centrifugation through 5.7 mol/L cesium chloride
step gradient.)’ Samples were enriched for poly A-containing RNA
by chromatography over oligo(dT) cellulose.” Aliquots were sizefractionated by electrophoresis through 1% agarose-formaldehyde
gels. The RNA was transferred to Genescreen (New England
Nuclear) and prehybridized at 42°C for 1 hour in 50% deionized
formamide, 0.5% SDS, 2X PIPES-NaC1-EDTA buffer, and 0.1
mg/mL salmon sperm DNA. The M-CSF probe was nick-translated
and labeled with 32P-dCTP(Amersham, Arlington Heights, IL) to a
specific activity of 1 x lo8 c p d k g DNA. Probe, 2 x lo7cpm, was
added to 20 mL of prehybridization solution and the blot was
hybridized for 16 hours at 42°C. Blots were washed sequentially
four times each in 2X SSC (1X SSC = 0.15 mol/L NaCV0.015 mol/L
sodium citrate, pH 7.4), 0.1% SDS at 22°C and 6 5 T , and 0.1X SSC,
0.1% SDS at 22°C for 15 minutes. Autoradiography was performed
with x-ray film and intensifying screens at -70°C.
Preparation of conditioned medium. Cells were seeded into
flasks and allowed to reach confluency in complete medium. Serum
containing medium was removed, and cells were washed once and
incubated in serum-free Fischer’s medium for 24 hours to eliminate residual contaminating serum. This medium was discarded
and replaced with fresh serum-free medium with or without PDGF
and bFGF. After a 3-hour incubation period at 37”C, the media
was removed, cells washed, and fresh serum-free medium added.
Cell-free supernatants were collected after an additional 8 hours,
sterile filtered (0.45 km), and stored at -20°C. In an additional
experiment, the effect of TNF on M-CSF secretion was examined
after 8 and 24 hours. Assays for CSF activity were performed on
aliquots of unconcentrated supernatants.
Radioimmunoassayfor M-CSF. M-CSF activity was quantitated
using a competitive radioimmunoassay developed by Stanley?’
Units are defined by an in vitro murine clonal assay, where 1 U
(0.44 fmol of M-CSF protein) is the amount of M-CSF required to
produce one colony from 7.5 x lo4 marrow cells plated in agar
culture. Results are expressed in units per lo6cells.
RESULTS
Growthfactor stimulation of L3H]-thymidineincolporation
and cell growth. Quiescent TC-1 stromal cells in culture
were assayed for their ability to incorporate [’HI-TdR into
DNA after exposure to PDGF or bFGF. As shown in Fig 1,
both PDGF and bFGF stimulated DNA synthesis in a
concentration-dependent manner. PDGF was a slightly
more potent mitogen than bFGF. Maximum stimulation of
[3H]-TdRincorporation into DNAof TC-1 cells occurred in
response to 10 ng/mL of PDGF or bFGF. In several
experiments, the fold stimulation varied from twofold to
fivefold with 10 ng/mL of PDGF or bFGF. In contrast to
PDGF and bFGF, incubation with TNF (1 to 25 ng/mL) did
not increase DNA synthesis. Time-course experiments (Fig
2) demonstrate that the addition of PDGF or bFGF
induced a progressive increase in [3H]-TdR incorporation
into DNA at 12 hours, reaching a peak effect at 24 hours for
PDGF and 32 hours for bFGF. DNA synthesis did not
increase over time in cultures treated with 10 ng/mL of TNF
(Fig 2). The stimulation of DNA synthesis by PDGF and
bFGF was confirmed by autoradiographic analysis and
determination of the labeling index as shown in Table 1.
There was a significant increase in the labeling index
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105
GROWTH FACTORS AND MURINE STROMA
Table 1. Effect of Peptide Growth Factors on DNA Synthesis of TC-1
Cells Measured by Autoradiography
Labeling Index
Condition
% o f Labeled Nuclei
Control
PDGF 1 ng/mL
PDGF 10 ng/mL
bFGF 1 ng/mL
bFGF 10 ng/mL
10% FCS
0.6
7.0(11)
69.8 (116)
14.8 (25)
42.2 (70)
19.7 (33)
Cells were plated onto 4-chamber Lab-Tek slides at a density of 1 x
IO4cells/chamberin Fischer's medium with 17% serum. Confluent cells
were made quiescent in medium containing 1% serum for 48 hours and
then incubated with PDGF or bFGF for 24 hours. 13V]-thymidine, 1.0
pCi/mL, was added at the time of growth factor addition. At the end of
the 24hour incubation period, cells were fixed and developed. Four
hundred cells per each incubation condition were counted and the
percent of labeled nuclei (labeling index) was determined. Numbers in
/
I
brackets represent fold stimulation.
= fv
1
1
0 1
*
I
5
IO
25
(ng/mL)
Fig 1. Dose-response curve for the effect of peptide growth
factors, PDGF and bFGF, on TC-1 stromal cell DNA synthesis. Cells
were plated in 24-well dishes at 5 x IO'cells/well in Fischer's medium
with 17% FCS. At confluence, cells were made quiescent by incubation in Fischer's medium with 1% serum for 48 hours. Growth factors
were then added and cells were simultaneously pulsed with ['HIthymidine (1.0 pCi/mL) for 24 hours. Control wells were incubated
with medium containing 1% serum alone. [3H]-thymidine incorporation into DNA was measured as trichloroacetic acid (TCA)-precipitable material. Each point represents the mean of data tested in
duplicate or triplicate wells.
-
n
E!
x
Ilr
IO -
E 9Q
0
Y
8-
\
LNF-a
7
Control
A
OLCY I>
I
I
J
24
36
48
Hours
Fig 2. Time course for the effect of PDGF and bFGF on TC-1
stromal cell DNA synthesis. Cells were plated and made quiescent as
described in the legend t o Fig 1. PDGF ( I O ng/mL) or bFGF (10 ng/mL]
was then added and cells were pulsed with ['HI-thymidine (1.0
pCi/mL) for 4 hours before harvesting at the indicated time points.
Data are expressed as percent change from control wells incubated
without growth factors at each time point. Each condition was tested
in quadruplicate. Representative of two separate experiments.
(116-fold increase for PDGF and 70-fold increase for
bFGF) when cells were incubated with the concentration of
PDGF and bFGF (10 ng/mL) that was shown to cause
maximal [3H]-TdRincorporation into DNA.
We also confirmed that the enhanced [3H]-TdR incorporation into DNA is associated with cell proliferation. Table
2 shows stromal cell growth in 1% serum or 1% serum with
10 ng/mL of PDGF or bFGF. Cell number increased after 3
and 7 days of exposure to PDGF and bFGF. As compared
with controls, cell number was not affected when cultures
were incubated in the presence of TNF.
Constitutive expression of mRNA encoding M-CSF gene in
TC-1 stromal cell line. Because TC-1 cells have been
shown to constitutively secrete M-CSF into conditioned
medium, we evaluated basal M-CSF gene expression in
cells grown in complete medium. TC-1-C-11 cells, which are
similar to TC-1, were also evaluated? As shown in Fig 3, a
Northern blot containing total and poly(A)+RNA isolated
from each cell line demonstrates two major hybridizing
species of about 4.4 and 2.2 kb and a less-abundant species
of about 1.4 kb. A similar pattern of transcript hybridization
was observed in mouse L cells that were used as a control.
Stimulation of M-CSF mRNA by peptide growth factors.
To determine if PDGF and bFGF stimulate the expression
of mRNA encoding M-CSF, time-course experiments were
Table 2. Effect of Peptide Growth Factors on the Growth of TC-1
Stromal Cells
Cell Number x lo4
Control
PDGF
bFGF
Day 3
Day 7
7.8 2 .3
10.9 2 .4
10.1 f .4
5.1 2 .5
13.3 2 .6
9.6 f 1
Cells were plated in medium with 17% serum and after 24 hours they
were placed in Fischer's medium with 1%serum and incubated with 10
ng/mL PDGF or 10 ng/mL bFGF. On day 3, triplicate wells from each
condition were trypsinized and counted using a Coulter counter (Coulter,
Hialeah, FL). Remaining wells had their media replaced with fresh
medium without (control) or with growth factors. Cell count at day 0
was 7.3 f .2 x 104/well(mean t SE). Data from two separate experiments, each performed in triplicate wells.
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ABBOUO AND RNZANI
Poly(A)+ RNA
c
c
1
I
0
v
I
I
0
0
t
I-
--c.
-
.-
-
M-CSF
m3. " w m b k t a M ) y r h
d M-CSF mRNA comtkuttvely
I
c
4.4
2.21.4-
pcrformcd using quicsccnt and PDGF- or hFGF-trcatcd
cclls. Figurc 4 shows a clcar. low-intcnsity signal in unstimulatcd cclls. F A p u r c of TC-I cclls to an optimal dcm o f
PDGF (IO n@mL) markcdly incrcascd the stcadv-statc
lcvcls of M-CSF mRNA by 1 hour. with a pcak cffcct at 3
hours subsiding to ncar-basal lcvcls by 16 hours. In rcsponsc to 1 0 n@mLof hFGF. induction of M-CSF mRNA
lcvcls was sccn within 2 hours. rcaching a F i i k cficct hy 6 to
I2 hours. and thc incrcascd mRNA lcvcl~pcrsistcd for I2
to 14 hours. T N F a also incrciiscd thc lcv~lof M-CSF
mRNA in TC-I cclls with a pcak cffcct occurring hy I to 1
hours. In all cxpcrimcnts. thc rclativc ahundancc of thc
thrcc M-CSF m R N A transcripts did not changc aftcr
induction. To dctcrminc thc spccificity of thc clfccts of
thcsc pcptidcs for M-CSF mRNA. blots wcrc hoilcd to
rcmwc M-CXF pmbc and rchyhridizcd to an a-tubulin
cDNA probc. Thcrc is littlc if any variation in tubulin
mRNA lcvcls. suggcsting that thc rcspcmsc in M - O F
mRNA is spccific and does not simply rcflcct a glohiil
incrcasc in total RNA.
To dctcrminc if thc incrcascd cxprcssion of M-CSF
m R N A is asuxiatcd with cnhanccd sccrction of thc corrcspconding protcin. wc uscd a scnsitivc radioimmunoassay to
dctcct M-CSF activity in both quicsccnt and PDGF- or
hFGF-stimuliitcd stromal cclls. Conditioncd medium collcctcd from quicsccnt stmmiil cclls ccmtiiincd 370 U/lV
cclls of M-CSF. Whcn cclls wcrc incuhatcd with 1 0 n@mL
of cithcr PDGF or hFGF. M-CSF sccrction incrcascd at 8
hours to 621 iind 644 U/l(rcclls. rcspcctivcly. Incubation of
TC-1 cclls with I O n@mL of TNF i i k ) incrcascd MI-CSF
sccrction fmm 447 to 697 UIIV cclls aftcr 8 hours and fmm
1.104 to 2.872 Ull(rcclls aftcr 24 hours.
DISCUSSION
Thc prcscnt study dcmonstrstcs that PDGF and bFGF.
hut not T N F a . arc potcnt mitgcns for murinc stromal
cclls and that thcsc pcptidcs markedly stimulatc thc cxprcs-
-5.I
- 4.4
-2.4
- 2.0
- I .4
exprnwd in TC-1 M d TC-14-11
stromal celh. FHtnn mkrogrmn
Of (01.1 CdlUl8r RNA and 5 pg Of
polv(A)'RNA from confluent cells
m r e fr8aionat.d on an agarow
0.1. Also shown is a blot containing m o u u L.cell WCSF mRNA
as 8 control. Blots were hybridI2.d with nkt.118Ml81.d M-CSF
cONA p r o k . N u m k r e d columns
represent sire in kilob88es (kbl
of RNA standards run in the u m e
gels M-CSF p r o k detects three
tran8cripts of 4.4. 2.2. and 1.4 kb
that approximate those transcripta e x p r n u d in mouse L
cells
sion o f m R N A cnadinp for M-CSF iind sccrction of thc
protcin. Thc mitogenic cffcct of PIIGF and hFGF on thc
murinc TC-I stromiil cclls was dtxumcntcd hy cnhanccd
DNA synthcsis. incrcasc in the labcling indcx hy autoradiography. and incrcaw in ccll grtwth in rcspcmsc to each
pcptidc. I t is wcll known thiit thc major sourcc o f PDGF is
thc a granulcs of pli~tclct~
iind thiit iictivatcd montxytcs.
cndothcliiil cclls. and fihmhlasts rclcasc PDGF and cxprcss
PDGF mRWAs that c n d c thc A andfor R chains of thc
PDGF molccuIc.*'H"Although thc prccisc role of PDGF
and hFGF in normal hematopoiesis remains to bc dctcrmincd. rcccnt studics havc sticnvn thiit PDGF stimuliitcs
the growth o f crythroid progenitors."" Dclwichc ct iily'
suggcstcd thiit this cficct of P I X F is mcdiatcd through two
acccswry ccll populations. fibroblasts and smwth musclc
cells. hut not cndothclial cclls or mitCn)phiigcS. Michiilcvicz
ct al." '' using a highly cnrichcd carly hone marrow population. found that P I X F dircctly stimuliitcd mixcd crythroidmycloid ccdonyforming units in this fraction. although an
indircct cficct through acccswry cclls was not cxcludcd.
Prcviousstudicshavc shown that PDGF is a potcnt mitcgcn
rind that it incrcascs II,-I. IL-6. and G-CSF mRNA trmscripts in human stromal cclls."" Rcccntly. hFGF has bccn
shown to cnhancc thc gmwth of human stromal cclls in
long-tcrm culture.* It also intcracts syncrgisticallv with
other CSFs, such as IL-3 and GM-CSF. to stimulatc thc
growth of carly human hcmatopcoictic prqcnitors." Our
linding raiscs thc possibility thiit hFGF rclcascd by activated macrophapcs miiy influence progcnitor ccll growth
indircctlyvia accessory cclls. I t is also likely that PDGF and
hFGF piirticipatc in thc hcmatopoictic rcspmsc to inflammat ion.
Thc cxprcssion of murinc M-CSF spccific transcripts in
TC- I cclls using total or poly A-cnrichcd ccllular RNA and
M-CSF activity is in agrccmcnt with a prcvious r c p n
dcmonstrating thc constitutivc rclcasc of M-CSF protcin
into medium by thcsc cells." Thc hybridization pattern
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GROWTH FACTORS AND W I N E STROMA
107
A g l . fwd.u-dllWrJF-bvp.p((d.gmw(h-.
"blot
8 f " d tobl RNA (15 pqp.r lnwl d Tc.1 m T a n D l
cr(h.hducckn of M.CSF "A o r p "
b IA) POW (10 ng/mL).
18) bFOF (10 ng/mLI. ond (C) TNF- (10 ng/mL). C o d h m t TC-1 c d h
wwo nud.q u W b pl.clng th.m orom(gM in 1.. sewn. PDGF.
bFOF. of TNFQ w n thorn .dd.d md eolh Incubated 101 tho indk4t.d
ttnW pohm Cytoplnmk RNA w n thm b.ol4t.d from tho wlls and
h*a(dkatCOn w n podomwd n d.rctlkd in the kg.nd to Fig 3.
comfol ~OIWS to RNA 1-1.d
ir~m
a i l s h r m ~ "~0t d
lfi 1%
sewn abne. Altu m l r o t k n with th. M-CSFcWA.th. w o k w n
runowd by b o h g md o u h blot w n rbhybddlrd with an dubulh,
c o w prob..
showing t hrcc clear hands of 4.4.2.2. and I .4 kh is similar to
that found in mousc I, cclls." Rcccnt studies havc addrcsscd the regutittion of M-CSF in honc m i i r m stromal
cclls. hut with conflicting rcsults. IL-I has hccn shown to
inclucc thc production of M-C'SF by human long-term
stromal ccll cultures.' Ihwcvcr. Gimhlc CI at.' using niurinc
stmmal cclls. w r c unahlc t o shcnv a significant change in
thc cxprcssion of M-C'SF mRNA hy a varictv o f cytokincs,
including 11.-1 and TSF. Incluction of M-C'SF mRNA has
also rcccntlv k e n rcp)nctl in 3T3 fihrohlasts wing high
Concentrations of thc puriliccl AR iwform o f PM;F."
Ikcauw both PlXiF and hFGF wcrc mitogenic to Tc-I.
an impwtant issue riiiscd hy thcsc findinp i\ whether the
cffcct of thcsc two pcptidcs on M-CSFmKNA rcprcscnts a
spccific sign;iling pathway or is rclatcd t o DNA synthcsis
and cell prcnvth. 1°F-a at a conccntration thnt markcdlv
incrcasccl M-C'SF lcvcls had no clicct on DNA synthesis.
suggesting that thc stimulatory clfcct of this cytokinc on
M-<'SFmRNA is not relatcd to cell growth. Thc unclcrlving
mechanism for thc cfTcct of P M i F and hFGF on IM-CSl:
mRNA is not known. While 1hi.i work was in progress,
FalkcnhurgCI al" rcponcd that PIXIF. hFGF. or comhinitlions of pcptidc pm-th f;icton that stimulatc proliferation
of murinc IOTE 1ihn)hlast cclls alw intlucc M-CSF mRNA
cxprcssion. Thc investigators suppcstctl that M-CXFcxprcssion mrrclatcs with thc pro1ifcr;itivc state of thc cclls. 11.-I.
on the othcr hand. which docs not appear t o havc a
mitqcnic cffcct in thmc cells. also stimulated M-C'SF
mRNA. similar to our lindings with TPIF. Thc prccisc mlc
and ;issociation of ccll-cycle cvcnts to thc regulation of
M-CSFmRNA rcmains t o hc dctcrmincd.
It i\likely that the rclcitw of M-CSF from stromal cells
mav act a\ r pmitivc fccdhack mcchanism to further
enhance thc pnxluction of moncrcytc PDGF. hFGF. and
other cytokines prcwitlinga pmitivc sign;il that amplilies thc
hcmatoprictic rcsp)nw to infl;immation. Thc rcccnt o k r vation that murinc erythroid cclls rclcasc P ~ X I;tctivity
F
in
vitro and in vivc).'"' taken tqcthcr with our prcscnt findings.
expands the mlc of Icrally prculucccd P M i F ;tnd pcrh;ip
ot hcr polypcpticlc growth f;tctors in regulatinghcmatop)icsix. Morccwcr. the potent stimulatory clrcct of PDGF and
hFGFon M-C'SFmRNA in TC- Istromal cclls s u ~ c s t that
s
thew cclls may pmvidc a uscful modcl t o study thc d l u l i i r
mechanismsof M-C'SFgcnc rcpulation.
ACKNOWLEDGMENT
The authorr thank Dr L R . Stanley for the M-CSFdeterminaI b n and Dr 1lanna AMrmd fm his amtinuour rupporl and advice.
We alu, ackncwiledgc Mclergarct Ciorman for e x p d technical
assistance nncl Iknina I k l r o n fcn typing thc manuu-ript.
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