1. No category

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1990 75: 1780-1787
Human colony-stimulating factor 1 (CSF-1) receptor confers CSF-1
responsiveness to interleukin-3-dependent 32DC13 mouse myeloid
cells and abrogates differentiation in response to granulocyte CSF
J Kato and CJ Sherr
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Human Colony-Stimulating Factor 1 (CSF-1) Receptor Confers CSF-1
Responsiveness to Interleukin-3-Dependent 32DCL3 Mouse Myeloid Cells and
Abrogates Differentiation in Response to Granulocyte CSF
By Jun-ya Kat0 and Charles J. Sherr
Interleukin-3 (IL-3)-dependent mouse myeloid 32DC13 cells
differentiate to neutrophils in response to granulocyte
colony-stimulating factor (G-CSF). Introduction of the human c-fms gene, which encodes the receptor for CSF-1,
into 32DC13 cells gave rise to variants that were able to
proliferate in medium containing either ‘murine IL-3 or
human recombinant CSF-1, but were unable to differentiate to granulocytes in response to G-CSF. Unlike parental
32CD13 cells, CSF-1 -responsive derivatives expressed nonspecific esterase when grown in CSF-1, but did not exhibit
many other morphologic, immunologic, or functional properties of mononuclear phagocyte differentiation, or express murine CSF-1 receptors. Accelerated turnover of the
human CSF-1 receptor was observed in response to CSF-1
and phorbol esters, but not after stimulation with IL-3 or
bacterial lipopolysaccharide. Although both CSF-1 and IL-3
induced tyrosine phosphorylation of heterologous substrates in the dually responsive cells, differences in the
patterns of substrate phosphorylation were observed in
response to the two hematopoietins. We conclude that
expression of the human CSF-1 receptor in 32DC13 cells
not only induces CSF-1 responsiveness, but alters its
phenotype in a way that prohibits granulocyte differentiation.
0 1990 by The American Society of Hematology.
C
sets of genes that independently contribute to each of these
activities. In support of this hypothesis, the effects of CSF-1
on cell proliferation and survival can be dissociated, since
submitogenic doses of CSF-1 can maintain the viability of
nonproliferating bone marrow-derived macrophages that
become growth arrested in the early GI phase of the cell
cycle. lo^'
An unresolved issue concerns the extent to which the
CSF-1R tyrosine kinase modulates the activity of genes that
determine different stages of mononuclear phagocyte differentiation. To approach this problem in a model system, we
introduced c-fms cDNA encoding human CSF- 1R1’ into
mouse 32DC13 cells, which self-renew in IL-3 but terminally
differentiate to neutrophils in response to G-CSF.13s’4Populations of CSF- 1R-bearing cells were obtained that proliferated in either IL-3 or CSF-1, expressed monocyte esterases,
and failed to differentiate in response to G-CSF. Our results
suggest that CSF-1R both positively and negatively regulates the expression of gene products affecting differentiation
within the monocyte and granulocyte lineages.
OMMITMENT OF immature myeloid cells to differentiate within the granulocyte and mononuclear phagocyte lineages is accompanied by alterations in expression of
receptors for different hematopoietic growth factors. Immature cells that respond to pluripoietins such as interleukin-3
(IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) become progressively more sensitive to the
actions of the lineage-specific growth factors, G-CSF, and
M-CSF or CSF-1, as they mature within the granulocyte or
macrophage lineages, respectively.’ Expression of the CSF- 1
receptor (CSF-1R) is first detected in mononuclear phagocyte precursors and increases as the cells
whereas
such cells exhibit reduced responsiveness to GM-CSF and
IL-3.’.4-6
The mechanisms by which the IL-3, GM-CSF, and
G-CSF receptors transduce intracellular signals remain
poorly understood. However, CSF- 1R exhibits an intrinsic
protein tyrosine kinase activity which, when activated by
ligand binding, triggers the phosphorylation of a series of
protein substrates, at least some of which relay intracellular
signals that induce CSF- 1-responsive genes.’ Because
CSF- 1 supports the proliferation, differentiation, and survival of cells of the mononuclear phagocyte
it
seems likely that its pleiotropic effects are mediated through
From the Howard Hughes Medical Institute, Department of
Tumor Cell Biology. St Jude Children’s Research Hospital; and
Department of Biochemistry, University of Tennessee College of
Medicine. Memphis.
Submitted November 17, 1989; accepted January 11,1990.
Supported by the Howard Hughes Medical Institute, and by
Cancer Center CORE Grant (POI -CA21765) to S t Jude Children’s
Research Hospital.
Address reprint requests to Charles J. Sherr, MD, PhD. Howard
Hughes Medical Institute. Department of Tumor Cell Biology. St
Jude Children’s Research Hospital, 332 N Lauderdale. Memphis,
TN 381 05.
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 1990 by The American Society of Hematology.
0006-4971/90/7509-000l S3.00/0
1780
‘
MATERIALS AND METHODS
Cell lines and culture conditions. IL-3dependent 32DC13
cell^'^.'^ and CSF-ldependent BACl.2F5 macrophages” were
generously provided by Dr Giovanni Rovera (Wistar Institute,
Philadelphia, PA) and Dr E. Richard Stanley (Albert Einstein
College of Medicine, Bronx, NY), respectively. The cells were
maintained in Iscove’s medium containing 20% fetal calf serum
(HyClone, Logan, UT), glutamine, antibiotics, and requisite growth
factors (either 20% Wehi-3B cell conditioned medium as a source of
IL-3 or 25% L-cell conditioned medium as a source of murine
CSF-1). The human glioblastoma cell line, U87 MG, also provided
by Dr Giovanni Rovera, was grown in complete Iscove’s medium
without other growth factors and was used as a source of G-CSF.I6
A BamHI fragment containing the intact human c-fmscDNA”
was inserted in both the sense and antisense orientation into an
inducible expression vector’’ containing four tandem upstream
copies of a metal response element,19 the &globin TATA box 5’ of
the BamHI cloning site, and a simian virus 40 polyadenylation signal
downstream of the inserted gene. The c-fms plasmids were each
electroporatedl’ into 32DC13 cells with a second plasmid (pSV2ne0)~’
that confers resistance to the antibiotic G418 (Geneticin, Sigma
Chemicals, St Louis, MO). Electroporated cells were seeded at 3 x
IOscells per well into 24-well multiplate culture dishes, and selected
Blood, Vol 75, N o 9 (May 1). 1990: pp 1780-1787
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REPROGRAMMING THE CSF-1 RESPONSE
for 3 weeks in complete medium containing IL-3 and 800 pg/mL
G418. The medium was then supplemented with heavy metals
(1 x IO-, mol/L ZnSO, and 5 x IO-’ mol/L CdSO,), and selection
was continued in the presence of IL-3 and G418 for 2 more weeks.
Equal aliquots of cells from G418-resistant cultures electroporated with the c-fms gene in sense orientation were pooled, and cells
expressing human CSF-1R were selected by fluorescence-activated
cell sorting using a monoclonal antibody (MoAb) to the human
receptor.” The sorted CSF-1R-positive population (designated
32D[R+] cells) was maintained in medium containing IL-3. In
parallel, cells from each independentculture were grown in complete
medium supplemented with 2,000 U/mL (0.9 nmol/L) of human
recombinant CSF- 1. Seven cultures yielding CSF- 1-responsive cells
were obtained, three of which (designated 32D[R+]Cl.l, C1.2, and
C1.3) were characterized in detail.
Other analytical methods. Cells in suspension culture were
concentrated on slides by cytocentrifugation, fixed in cold 9.25%
formalin, 45% acetone in sodium phosphate buffer, pH 6.6, and
stained for 1 hour at room temperature in an esterase reaction
mixture (pH 6.3) containing 50 pg/mL a-naphthyl butyrate as a
substrate.,, In some reactions, 1.25 mg/mL sodium fluoride was
added to the reaction mixture to inhibit nonspecific esterase activity.
Procedures for metabolic labeling with [35S]methionine,cell lysis,
immunoprecipitation,polyacrylamide gel electraphore~is,~’
and immunoblotting with antiserum to phosphotyrosineZ4are described in
detail in the referencescited.
Special biologic reagents. Purified murine IL-3 was supplied by
Dr James Ihle (St Jude Children’s Research Hospital, Memphis,
TN) and human recombinant CSF-I was obtained from Dr Steven
Clark (GeneticsInstitute,Cambridge,MA). 12-0-tetradecanoylphorbol-13-acetate (TPA), bacterial lipopolysaccharide (LPS), prostaglandin E, (PGE,), 5-azacytidine (all from Sigma Chemicals),
1,25-dihydroxyvitamin D3, and leukotriene B4 (LTB4) (both from
Biomol Research Laboratories, Plymouth Meeting, PA), and mouse
recombinant GM-CSF (Genzyme Corp, Boston, MA) were obtainedfrom the indicated vendors. Production of MoAbs to human CSF1R2’and affinity-purified rabbit antibodies to phosphotyrosineZ4was
described previously.
Preparation of rabbit antiserum to CSF-IR. An NcoI fragment
(nucleotides 2271 to 3280)” representing the 3’ end of the human
c-fms gene, was inserted into a pBR322-based expression plasmid,
P S J H ~ ~after
, , ~ conversion of its unique ClaI and BamHI cloning
sites into NcoI and XhoI sites, respectively.26The resultant construct
contained the X pL promoter, and coded for a thermoinducible X
C,,-c-fms fusion protein of 37.5 Kd containing the carboxylterminal
340 amino acids of human CSF-1R. The plasmid was inserted into
Escherichia coli strain N4830, which contains a thermolabile pL
repressor. After culture at 42OC, the induced fusion protein was
purified by differential salt fractionation and electrophoresis on
denaturing gels, and the eluted polypeptide was used to immunize
rabbits.” The resulting antiserum to human CSF-1R crossreacts
with both murine CSF-IR and the feline v-fms oncogene product,
and supports their kinase activities in in vitro enzyme assays
performed with immune complexes.
1781
we introduced the human c-fms gene encoding CSF-1R into
these cells and tested their response to different hematopoietins.
Human c-fms cDNA, cloned into an inducible expression
vector containing a metal-responsive enhancer fused to the
P-globin promoter,’* was electroporated into 32DC13 cells
with pSV2ne0, a plasmid that confers resistance to the
antibiotic G418.*’ The electroporated cells were then divided
a t 3 x l o 5 cells per culture into a 24-well multiplate and
grown for 3 weeks in medium containing IL-3 and G418.
Twenty-one G418-resistant cultures were obtained and passaged for 2 additional weeks in medium containing IL-3 and
heavy metals to induce expression of human CSF-1R. Equal
aliquots from each of the 21 cultures were harvested and
pooled, and CSF-1 receptor-positive cells (here designated
32D[R+]) were purified by fluorescence-activated cell sorting (FACS) with an MoAb directed to an extracellular
epitope of human CSF- 1R.*’ These cells were continuously
maintained in medium containing IL-3. Under these conditions, 32D[R+] cells expressed moderate levels of human
CSF-lR, even in the absence of heavy metals (Fig l ) , and
receptor expression was not increased further when heavy
metals were added to the medium.
In parallel, cells from the 21 individual G418-resistant
cultures were transferred into medium containing human
recombinant CSF-1 and heavy metals, yielding seven cultures capable of continuous growth in CSF-I. By contrast, 24
cultures of 32DC13 cells electroporated with a plasmid
containing the c-fms gene in an anti-sense orientation together with pSV2neo yielded l l G418-resistant cultures,
none of which were able to grow in human CSF-1. Thus,
CSF- I-responsiveness appeared to depend on expression of
the transduced c-fms gene.
Of the seven CSF- 1-responsive cultures, three grew very
slowly, whereas one showed a factor-independent phenotype
and proliferated in the absence of either CSF-1 or IL-3. The
remaining three cultures (designated 32D[R+]Cl.l, C1.2,
and C1.3) were dependent on either human CSF-1 or murine
RESULTS
Transduction of human CSF-IR into IL-3-responsive
32DC13 cells. The mouse myeloid cell line, 32DC13, is
dependent on IL-3 for proliferation and survival, but terminally differentiates into mature granulocytes when IL-3 is
removed from the culture medium and is replaced by
G-CSF.”,I4 To determine whether 32DC13 cells could be
reprogrammed to proliferate in CSF-1 and whether stimulation by CSF-1 would affect their pattern of differentiation,
LOG FLUORESCENCE INTENSITY
Fig 1. Flow cytometric analysis of 32DC13 cells transfected
32D[R+] cells
with human c-fms. Parental 32DC13 cells (-----I.
grown in IL-3 (.-.--I.
and 32D[R+]Cl.l cells selected for growth
in CSF-1 (-4were studied using an MoAb reactive to human
CSF-1R?’ Background fluorescence with an isotypematched control antibody is also indicated (.
).
.- ..
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KAT0 AND SHERR
1782
IL-3 for survival and proliferation, but died in the absence of
either factor. When these CSF-1-responsive cell lines were
analyzed by FACS for expression of human CSF-lR, they
expressed significantly higher levels of the receptor than
pooled 32D[R+] cells grown in IL-3 (Fig 1). These levels of
CSF-1R are equivalent to those detected on murine
BAC1.2F5 macrophages (about 1 x lo5 CSF-1 binding
sites/cell). Although the experimental protocol prohibited an
estimation of their frequency, the results suggested that only
those cells that expressed high levels of human CSF-1 R
selectively grew out in response to CSF-1. Cells from the one
factor-independent culture also exhibited very high levels of
CSF-1R expression (data not shown), consistent with previous results suggesting that overexpression of CSF-1 R in
immature myeloid cells may contribute to a factor-independent phenotype.'8
Growth and differentiation of CSF-1-responsive cells.
Subsequent experiments were performed with the three
CSF-1-responsive cell lines and with single cell subclones of
each. Because similar data were obtained with each of these
cell lines, only representative data with 32D[R+]Cl.l cells
are shown below. Figure 2 shows growth curves under
different conditions for parental 32DC13 cells (A) and for a
CSF- 1-responsive clone (B). The parental cells proliferated
only in the presence of IL-3. When transferred to medium
containing human recombinant CSF-1, their viability declined rapidly, and no CSF-l-responsive or factor-independent variants arose from cultures containing more than 5 x
lo6 cells. In contrast, 32D[R+]Cl.l cells grew equally well
in response to murine IL-3, human CSF-1, or in medium
containing both growth factors, but died in the absence of
either. Although human CSF-1 can bind with high affinity to
both human and mouse CSF-lR, murine CSF-1 does not
bind to human CSF-1R or induce the growth of cells bearing
the human
As expected, murine CSF-1 did not
support the growth or survival of 32D[R+]Cl.l cells,
implying that they did not express murine CSF-1R (see
below) and that their growth in response to human CSF-1
was mediated by the transduced c-fms gene product.
When washed and transferred to medium containing
G-CSF, 32DC13 cells eventually stopped growing (Fig 2A)
and differentiated to granulocytes. Figure 3 contrasts the
morphology of the parental cells maintained in IL-3 (A) with
those cultured for 10 days in G-CSF (B) and illustrates the
conversion of immature myeloid cells to more mature granulocytes containing banded or segmented nuclei.14 In contrast,
the survival of CSF-1-responsive 32D[R+]Cl.1 cells was
not supported by G-CSF (Fig 2B). Although these cells could
be maintained for a somewhat longer period of time in
medium containing G-CSF than in unsupplemented medium, no viable cells remained after 10 days of G-CSF
treatment. Moreover, no morphologic evidence of granulocytic differentiation was observed in G-CSF-treated
32D[R+]Cl.l, C1.2, or C1.3 cultures maintained during
this period, nor did G-CSF inhibit their growth in response to
either IL-3 or CSF-1 (data not shown).
Because the CSF-1-responsive cell lines were unable to
differentiate to granulocytes in response to G-CSF, we
considered the possibility that CSF- 1 induced an alternative
genetic program leading toward monocytic maturation.
Therefore, the cells were assayed for production of nonspecific esterases as markers of monocytic differentiation. The
term nonspecific esterase is reserved for enzymes capable of
hydrolyzing simple esters of N-free alcohols and organic
acids. For those enzymes that use a-naphthyl butyrate as a
substrate, the reaction products are generally confined to
5
monocytes.22Parental 32DC13 cells lacked this activity (Fig
In
3A), as did those that had been induced to differentiate to
,
,
,
,
,
,
,
,
granulocytes (Fig 3B). CSF-1R-positive 32D[R+] cells that
X
0
1
2
3
4
5
6
7
8
9
10
u)
had never been cultured in CSF-1 contained occasional
= 10000
5000
B
nonspecific esterase-positive cells (Fig 3C), whereas
32D[R+]Cl.l cells maintained in human CSF-1 exhibited
1000
500
high levels of enzymatic activity (Fig 3D). When the latter
cells were washed, resuspended, and cultured for 2 more
100
50
weeks in medium containing IL-3, the cells remained highly
esterase-positive (Fig 3E), suggesting that induction of this
10
5
activity by CSF-1 was not readily reversible. The enzyme
was completely inhibited by addition of sodium fluoride to
1
0.5
the reaction buffer (Fig 3F), providing further evidence that
0
1
2
3
4
5
6
7
8
9
10
it represented a monocytic isoform.22
Days
Although the CSF- 1-responsive cell lines were esterasepositive,
they lacked many other features of monocytic
Fig 2. Growth of parental 32DC13 (A) and CSF-1-responsive
differentiation. The cells remained nonadherent, were non32D[R+]Cl.l cells (9) in different media. Cells seeded at the
phagocytic, could not present antigens to their respective
indicated densities in T26 flasks were grown in unsupplemented
Iscove's complete medium ( 4 ) or in media containing IL-3 (0). T-cell hybridomas, were unable to support an antibodyCSF-1 (A),G-CSF (m), or IL-3 plus CSF-1 (0).Exponentially
dependent cytotoxic response to sheep erythrocytes, and
proliferating cultures were counted, depopulated, and fed every 2
expressed only low levels of the MAC-1 antigen3' equivalent
days. and the total cells were estimated from the growth of the
to those detected on parental 32DC13 cells (negative data
remaining population.
1
,.at\
3
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REPROGRAMMING THE C y - 1 RESPONSE
-
~
fornulin-owtono. mhml tor “dfk oatu8ao uring cr-~phthyl-ate
as
wbstrate. and aowtus”with
ha”ory)(n. Tho panoh.haw 320C13colh maint8id in k-3 (A): 32OC13 colh grown for 10In ECSF (8):32D(R+] colh maintobad
in IL-3 (C): 3 2 q R + lCl.1 c.(h maint8icHd in CSF-1 (0):3 2 q R + Kl.1 cdh maintained in CSF-1and then washed 8nd grown for 2 vmlu in
medium containing IL-3 (El: and the u m o c d l s as in panmi 0 stained tor ..to” in tho pr...(~. of d i m fluorid. (FI.
kr(hr.d
not shown). Treatment of these cells with other potential
inducing agents including GM-CSF, LPS. TPA. PGE,.
LTM, I .25-dihydroxyvitamin D3. or 5-azacytidine did not
induce monocytic maturation.
32D/R+lCI.I cells do not express murine CSF-IR.
The m a t typical marker of maturation in the mononuclear
phagocyte lineage is expression of CSF-IR itself.*~’.’.’’The
inability of murine CSF-I to support the proliferation of
32D[R+]Cl.l. C1.2.and C1.3cellssuggested that they did
not express murine CSF-IR. To confirm these results.
32DC13,32D[R+]. and 32D[R+]Cl.l cells were metabolically labeled with [”S]methionine,and human CSF-I R was
immunoprecipitated from detergent lysates using an MoAb
that does not crossreact with murine CSF-I R.” The cleared
lysates were then reacted with a polyvalent rabbit antiserum
raised to a human c-fms polypeptide that crossreactswith the
murine receptor. As a positive control. lysates of metabolically labeled murine BAC I .2F5 macrophages. which express
high levels of the mouse CSF- I receptor.“’.” were analyzed in
parallel. All immunoprecipitates were denatured and *parated on polyacrylamide gels containing sodium dodecyl
sulfate, and receptors were detected by autoradiography of
the dried slab gels. Under these labeling conditions. both the
immature intracellular form (about 130 Kd) and mature cell
surface form (about 165 Kd) of CSF-IR are detected; the
two forms differ solely in their composition of asparaginelinked oligosaccharidechains.’
Figure 4 (lanes I and 5 ) shows that parental 32DC13 cells
did not express CSF- 1 R. As expacted from FACS analysis
(Fig I). 32D[R + ] c e l l s e x p r d significantlylower levelsof
human CSF-IR (lane 2) than 32D[R+]CI.I cells (lane 3).
whereas both cell lines failed to express murine CSF-IR
(lanes 6 and 7). The MoAb to human CSF-I R does not react
with the murine receptor e x p r d in BACl.2F5 cells (lane
4). but the latter species was readily detected with the
polyvalent rabbit antiserum (lane 8). Again. by these criteria, the levels of human CSF-l R synthesis in 32D[R + IC1 . I
cells were similar to those observed for the murine rcceptor in
BACI.2FS macrophages. However, the molecular mass of
human CSF-I R in 32DC13 cell derivatives was greater than
that of the murine receptor in BAC1.2FS cells, probably
reflccting cell-spacificdilferencesin the processing of oligosaccharide chains. Similar results were previously observed for
v-fms gene products expresscd in murine myeloid“ and
macrophage’* cell lines, respectively.
Human CSF-IR is not transmodulated by IL-3. After
ligand binding. CSF-I R is rapidly internalized and degraded
in lysosomes (receptor downmodulation).’.’? Activators of
protein kinase C. such as the phorbol ester TPA. also induce
accelerated receptor turnover”.” by inducing a protease that
cleaves CSF-I R near its transmembrane segment; this process (receptor transmodulation) results in the release of the
receptor ligand-binding domain from the cell and concomitant internali7ation of the SO-Kd tyrosine kinasedomain that
is degraded intracellularly.p Treatment of macrophages
with bacterial lipopolysaccharideinduces transmodulationof
CSF-I R through the same mechanism.” Because IL-3 has
been reported to induce a loss of CSF-l binding sites from
mouse bone marrow cells.” we attempted to determine
whether it would similarly induce CSF-I R turnover in
32D[R+]Cl.l cellsthat expttssod bothclasscsof receptors.
32DIR+]C1.1 and BACI.2F5 cells labeled with
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
KAT0 AND SH€RR
1784
w
-
m
(m
d WF-111. S2OClJ (m
1 rd 6). sro(rr+] (m
2 rd 6). 32qR+)Cl.l
3 rd 7). rd MC11Fb
4. M.C.bd0
( I . m 4 w d b l ~ m o ~ ~ r r k h O ~ c n C ) l m L L ~ ~ f o r l . 6 h a M w d ) y . . d w k h ~ . A t n r r m a n l d
nw(.(. m
o
m-0
C
i
r
n lm"pr.dgturod*rt(h on MoAb tohunun C 8 F - l R I t " 1 ( h r W 4 1 . wd tho W o d ) y u t n m r o t h r ,
npr.dp(t.tod*rith~r.bbkWhwumu~wotirnrrichm*(rwC8F-lR~hrSchr~bl.hmurwcanp(.~ar*romh.d.drun*.d.
wd ..p.r.tod
on 7
gob contewq .odiun
.unotO. Tho po"d tho IIU1ur. Igpl66)and imcrutur. fOp130)
rocoptor @ycoprotolnsh BAC19FS mwrOgh.0.. llww 81 ond In 32OC13 cells tr~nsfoetodwith tho hunwn e - h gono (arrorrhwd.). as
m(( as th.porkkm d "Jw wdght n u r k r s Ln k l k b h o m . YO kdlccnod h tho nurgkn.
['%]methionine were either incubated in unsupplemented
maIiumorex.pocedtoCSF-I.TPA. LPS.or IL-3 for I hour
beforelysis. Human (32D[R+]Cl.l)or mowc(BACI.2FS)
receptors were then immunoprecipitated and resolved on
denaturing gels. Figure 5 shows that the mature cell surface
form of human CSF-IR (lane I ) was degraded after treatment of 32D[R+]Cl.l cells with CSF-I (lane 2) or TPA
(lane 3). but was not transmodulated by IL-3 (lane 4).
Similarly. murine CSF-IR expressed in BhCI.2F5 cells
(lane 6) showed aarleratcd turnover in response to CSF-I
(lane 7) and TPA (lane 8). but was U M ~ ~ C C by
I ~ IL-3
~
(lane
9). LPS transmodulated CSF-IR expressed in mouse macrophages (lane IO). but unexpectedly did not induce reccptor
turnover in 32DIR + IC I . I myeloid cells (lane 5). The
explanation for its di!Tercntial effect in the two cell typcs
remains unclear. although the simplest interpretation is that
immature myeloid cells lack a receptor for LPS.
Trrositw phmphorylurion induced 6y CSF-I und I L 3 .
Whereas CSF- I R functions as a protein tyrosine kinase:"
thestructureof the 11.-3 receptor and its mechanismofsignal
transduction remain unclear. Isfort et al" reported that
murine IL-3 binds to a 140-Kd protein that becomes p b
phorylated on tyrmine. suggesting that IL-3. like CSF-I.
might mediate its effms through the activation of a protein
tyrosine kinase. To determine whether CSF-I and IL-3
v
-
would induce the phosphorylation of similar substrata in
dually responsive cells. 32DC13. 3 2 D [ R + 1. and
32DIR+JCI.I cells were incubated in medium lacking
growth factors for 18 hours. and then were mtimulated for
10 minutes with high concentrations of IL-3 or CSF-I. Cell
lysates were pa rated on denaturing gels, transferred to
nitrocellulasc. and probcd with an antibody to phosphotymine. Important for the interpretationofthcscexperiments.
immunoblotting of total cell lysates with anti-phosphotymine is a relatively insensitive technique that only detects
the presence of major phosphotyrosine-containing polypcp
tides.
After IL-3 stimulation. two major phosphotyminccontainingbands were detected in each cell line (Fig 6. lanes
2.4. and 7). A protein of 140 Kd corresponded in mass to the
previously reported IL-3 binding proteinu; a second polypep
tide of 95 Kd showed an even greater signal intensity. In
contrast. CSF- I stimulation induced tyrosine phosphorylation of CSF-I R (about 165 Kd) as well as the appearance of
I IO-. 9% and 8SKd phosphoproteins (lane 8 ) . We also
observed a phosphoprotein of the same molecular weight as
CSF-IR in starved 32D[R+ ICl.1 cells (lane 6):this background level of receptor tyrosine phosphorylation was even
higher in the factor-independent variant (data not shown).
suggesting that high levels of receptor expression are accom-
-
F b 6.
rd
d CSF-lR. prJI( cuhtlm d sro(rr+)Cl.l orlr (Inw 1 thraudt 6) or MCl1Fb orlr
~lmno.6 thraug), 10) (WI. ).k(.d for 16 m(r*R..rrk),02m c l l d L-ps)mrctJorJn. rd harbtd Inbdlhg th.
pruur" for Bn.ddhtoru(2 hours. c.*.mn th.n orpaod for 8n .ddltbrwl hou to mnnd nwd(un ~hr
1 and 6 ) ff tonwdlvn
. u 9 9 ( w m d w k h 2 n n d l L -"b*wm
C8F-1fbOO2 rd 7):s x 10 " O l l L TPA (h0830nd8):2#) U l m l m ~ ( r w 1 1 - 31-4
and 9). OI 1 B a l d LPS fbOO 6 .nd10).CSf-1RW S i"unopr.dp)td wd W
N OS In
~ Fig 4: tho pork& d th.N 1 ~ CON
0 .Vtm
r m p m klndkotod h t h o r o n mugbn.
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REPRWRAWMING TU€ cslsl REspoNsE
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-3
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P n i d bY an increased basal level OfreceptOf kinase aaiGtY.
even in the abacno~of its ligand. Reincubation of the blots
with PbPhotYmine- but not PhasPbrine or PhasPbhm
onine. completely inhibited the ability of the antibody to
detect either CSF-1 R Or the I 1@.95-. and M-Kd P h P h m
wins (data not shown). Together. the results SU"I
that
lb-3 induces a diflerent pattern of protein tyrosine p h w
phorylation than d m the Csf- I R kinase. although one O f
the substrata (95 Kd) may be phasphorylated in r-ponse to
treatment by either factor.
DlSCUSSW
IL-3dependent mouse 32DC13 cclbdonot expressdetectable CSF-I receptors and cannot survive in medium containing murim CSF-I. Insertion of the human c-/" gene into
t h a e cells enabled them to proliferate in human recombinant
CSF-I without affecting their ability to respond to murine
IL-3. Thus. transduction of a macrophage-specific growth
fanor reccptor into immature myeloid cells is sutkient to
sensitize them to a growth factor that normally acts on more
mature cells of the mononuclear phagocyte lineage. The
ability of receptors of the protein tyrosine kinase gene family
to reprogram growl h factor responsiveness when ex&
outside their normal cellular context was not unexpected.
Introduction of the human c-fms gene into mouse NIH-3T3
fibroblasts dependent for growth on platelet-derived growth
factor and insulin"."or into IL-34ependent FDC-PI mouse
myeloid cells'"' enabled them to proliferate in CSF-I.
Similarly. introduction of the c-&R protooncogene encodingthecpidermalgnnwth factor(EGF) receptorinto32DC13
or F X - P I cells rendered them responsive to EGF.*'
Although transduction of the CSF-I and EGF receptor
gena into mouse myeloid cell lines could. in =me cases.
induce ligand-dependent shifts toward more mature
phenotypes."." the cells did not terminally differentiate.
Moreaver. insertion of constitutive oncogene axled tyrosine
kinases. such as ~ - t r h Rv-ubl."'
.~
v-src..' or v-jms." into
these same cell lines abrogated their IL3 depmdence but did
not induce differentiation and. in the case of 32DC13 cells.
inhibited granulocyte maturation in response to G-CSF.U
The growth of 32DlR + IC1 . I . CI .2. and CI .3 cells in CSF-I
induced nonspecific aterase activity and rendered the cells
unable to differentiate in response to G-CSF. Induction of
esterase activity was not readily reversible. since m l t i v a tion of the cells for 10 days in IL-3 did not lead to a
detectable diminution of enzymatic activity. One interpretation is that CSF-I induced partial differentiation toward
monocytes. leading concomitantly to a Ims of G-CSF mponsivenesr. However. the cells did not exprw other functional
or phenotypic markers of monocytic differentiation. including murine CSF-IR itself. nor could they be induced to
mature within the monocyte lineage by GM-CSF. LPS.
PGE,. LTM. TPA. vitamin D3. or 5-azacytidine. Thus.
although 3 2 x 1 3 cells remain competent to differentiate to
granulocytes. they may be inherently unable to complete the
alternative differentiation program characteristic of nann~l
mononucleer phagocytes.
Normal IL-3-responsive bone marrow progenitors are
committed to differentiate, and although more mature myeloid elements arising from them can be isdated. they cannot
be mainlaid in culture: In cont-1, the praasofatablhhing factor-dependent cell lines may select for the constitutive
expression of tran-hptional rrgulatory factors that inhibit
receptor-mediated signals for diflerentiation. Mouse leukemic cells containing rctwirally activated emyh.. and ~ d 1.' genes r e p m n t examples of immomlizd lines that
=in
lL.3depedent but are unable 10 differentiate.The
latter protooncogenes encode nuclear DNA-binding p m
teins that are likely to regulate gene expression. Evi-I is not
normally expressed in hematopoieticcells." but its activation
in myeloid cells interrupts their differentiation program
without abrogating their growth factor dependence. We
would predict that introduction of e/" into such cells might
render them CSF-I responsive without inducing complete
monocytic maturation. An additional caveat in interpreting
our experiments is that human CSF-IR m y not function
appropriately in mouse myeloid cells: eg. FDC-PI cells that
e x p d human CSF-I R s h o d no evidence of monocytic
differentiation," whereas the murine c-/mJ gene induced
monocytic features when the cells were propagated in CSFI .lo
A n a introduction of the human e/mrgem with flVzn0.
the levels of human CSF-IR expressed in a mixed popllation of receptor-positive 32DIR + I cells were relatively low.
and only 7 of 21 individual G4IR-resistanr cultures ultimately gave rise to cells that grew in CSF-I. In contrast.
32D(R+ lCl.1. C1.2. and C1.3 cells. selected for growth in
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KAT0 AND SHERR
1786
medium containing human recombinant CSF- 1, expressed
significantly more CSF-1R than cells passaged in IL-3, as
judged both by FACS analysis and by biosynthetic labeling
studies. Thus, only those transformants expressing high
levels of human CSF-1 R appeared to have had a proliferative
advantage when the cells were shifted to medium containing
CSF-1. In FDC-P1 cells, high levels of human CSF-1R
expression were also associated with the emergence of
factor-independent variants, which depended on CSF- 1Rmediated signals for ce1lgrowth.l8 Incontrast, 32D[R+]Cl.l,
C 1.2, and C 1.3 cells remained factor-dependent throughout
months of continuous passage in culture, as did single-cell
subclones derived from them, and only one factor-independent cell line was obtained. Based on the elevated basal levels
of receptor tryosine phosphorylation observed in these cells,
we presume that CSF-1R kinase activity can provide certain
variants with a selective growth advantage, even in the
absence of ligand.
Because 32D [R + ] C 1.1 cells remained dually responsive
to IL-3 and CSF-1, we were able to examine whether IL-3
treatment transmodulated the CSF-1 receptor by inducing
its accelerated degradation. We considered this possibility
because IL-3 treatment at 37OC can induce the rapid loss of
CSF-1 binding sites from normal mouse bone marrow cells
by an as yet ill-defined me~hanism.~'
Although phorbol esters
and other inducers of protein kinase C can transmodulate
CSF-1R by activating a protease that cleaves the receptor
near its membrane-spanning segment,34IL-3 treatment had
no effect on CSF-1R turnover in 32D[R+]Cl.l cells. Reciprocally, insertion of the constitutively active v-fms oncogene
into FDC-P1 cells, although able to confer factor-independence, did not affect the number or affinity of IL-3 binding
sites expressed at the cell surface.31TPA transmodulated
CSF-1R in both 32D[R+]Cl.l and BACl.2F5 cells, but
LPS induced CSF-1R turnover only in BAC1.2F5 macrophages. This implies that LPS does not mediate its effects by
directly activating protein kinase C, but rather, its active
moiety (lipid A) may bind to another receptor that is
differentially expressed in these two cell types.
Although the mechanisms of signal transduction by the
IL-3 receptor remain unclear, IL-3 has been shown to bind to
a 140-Kd cell surface glycoprotein and to stimulate its
phosphorylation on tyrosine residues.36 After stimulation of
32D[R+]Cl.l cells with IL-3, immunoblotting analyses
performed with antibodies to phosphotyrosine showed the
presence of a 140-Kd substrate as well as a second major
tyrosine phosphorylated species of 95 Kd. One or both of
these proteins might well represent components of the IL-3
receptor or, alternatively, substrates of an IL-3 receptorassociated tyrosine kinase. CSF- 1 treatment of the same cells
induced the appearance of different tyrosine phosphorylated
substrates, including human CSF-1R itself. However, like
IL-3, CSF-1 induced tyrosine phosphorylation of a 95-Kd
protein. Apart from their molecular masses, we have no
further biochemical evidence that the 95-Kd phosphoproteins observed in response to IL-3 and CSF-1 represent the
identical polypeptide. Therefore, although direct transmodulation of CSF-1R by IL-3 was not observed, our results do
not preclude "crosstalk" between the two receptor signaling
pathways.
ACKNOWLEDGMENT
We thank Drs Giovanni Rovera and Richard Stanley for murine
cell lines; Dr Steven Clark for human recombinant CSF-I; Dr
Richard A. Ashmun for performing Row cytometry;and Dr Martine
Roussel and Virgil Holder for preparing the c-fms expression vector
and antiserum to its product.
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