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Differential Regulation of Macrophage Differentiation in Response to
Leukemia Inhibitory Factor/Oncostatin-M/Interleukin-6:
The Effect of
Enforced Expression of the SCL Transcription Factor
By T. Tanigawa, N. Nicola, G.A. McArthur, A. Strasser, and C.G. Begley
The physiologic program of macrophagedifferentiation normally proceeds in a coordinated manner in response t o several different growth factors. Although the utilization of
common receptor subunits may explain in part overlapping
biologic functions, mechanismsby which unique actions are
mediated remain obscure. We examined growth factor-induced macrophagedifferentiation in M1 leukemia cellsthat
simultaneously display receptorsfor interleukin-6 (IL-61, leukemia inhibitory factor (LIF) andOncostatin-M (OSM). Differentiation induced by all three factors was associated with
decreased expressionof transcription factors myb and SCL,
increased expressionof macrophage markers, and suppression of proliferation. Cell lines were established in which
SCL expression was enforced. In theabsence of growth factors, cells were indistinguishable from parental cells. However, LIF (or 0SM)-induced macrophage differentiation was
perturbed; there was failure to undergo morphologic differentiation, disturbed expression of lysozyme andMacla. and
failure t o suppress proliferation. Surprisingly the perturbation of macrophage differentiation did not apply t o induced
expressionofmacrophage
colony-stimulating factor (MCSF) or granulocytecolony stimulating factor (G-CSF) receptors. This dissociation of elements normally coordinated in
a macrophage differentiation program applied at a clonal
level. Therewas no disturbance of IL-6induced macrophage
differentiation. These data directly implicate SCL in components of the macrophage differentiation program (suggesting that LIF receptor/gpltO heterodimersutilize an SCLinhibitable pathway while gp130 homodimers do not) and
demonstrate differential-regulation of components of the
mature macrophage phenotype.
0 1995 by The American Society of Hematology.
G
T-cell acute lymphoblastic leukemia (T-ALL).2’-23Despite
its involvement in T-ALL, SCL expression in hematopoietic
cells is normally restricted to progenitorhtem cell populations, erythroid cells, mast cells, and megakaryocytes,”-”
and endothelial cell^.'^ Furthermore SCL has been directly
implicated as a positive regulator of erythroid differentiat i ~ n ? ’ .Conversely,
~~
as K562 and M1 cells were induced to
differentiate along monocytic pathways, the levels of SCL
In this study we
mRNA and protein became
undete~table.’~~~’.~~
have used M1 cells in which SCL expression was enforced to
examine some of the elements that are normally coordinated
and contribute to a physiologic programof macrophage differentiation. We demonstrate that while some components
of a
normal macrophage differentiation program could proceed normally to completion,otherelementsweresignificantlyperturbed and this dissociation applied at a clonal level.
ROWTH AND DIFFERENTIATION of mammalian
cells is a highly ordered and tightly regulated process
that involves multiple changes in gene expression, finally
resulting in a cell that has acquired a highly specialized
function and usually lost its proliferative capability. The hematopoietic system provides one example whereby this process is constantly required to replenish short-lived mature
cells of multiple lineages from a pool of progenitor cells and
stem cells. A number of hematopoietic cell lines have been
established that provide useful models of hematopoietic differentiation, and of growth factor action and intracellular
signaling events. One such cell line is the murine M1 myeloid leukemia cell line’ that can be induced to differentiate
in response to the growth factors leukemia inhibitory factor
(LIF), interleukin-6 (IL-6) and Oncostatin-M (OSM).’“ The
commitment to terminal differentiation induced by these
growth factors is associated with typical morphologic and
phenotypic characteristics of mature macrophages and with
complete suppression of proliferative potential of clonogenic
cells.’ The receptor complexes through which these three
growth factors signal have been partially e l ~ c i d a t e d ~and
.~-~
in each case the signal transducer molecule gp130 forms an
important component of the receptor?.” There is also evidence that the initial intracellular signaling events are identical when LW or L - 6 act on M1 cell^,""^ although at least
in some situations these signaling pathways could be distingui~hed.”*’~
Although numerous markers of macrophage differentiation and activation have been defined and some of the
intracellular signaling molecules elucidated, little is known
about the process of differentiation-induction that is normally apparently inextricably coupled to suppression of proliferative potential and loss of clonogenicity.
The SCL gene product is a member of the helix-loophelix (HLH) class of transcription factors” that are known
to be involved in development and differentiation events in
a wide variety of species and tissues. SCL (also known as
T C L J and Tal-l)18,’9was first identified because of its
involvement in a human stem cell leukemia” and has subsequently been shown to be involved in up to 25% of human
Blood, Vol 85, No 2 (January 15). 1995: pp 379-390
MATERIALS AND METHODS
Enforced expression of SCL in MI cells. An SCL retrovirus was
constructed using the entire murine SCL coding region33introduced
From the Walter and Eliza Hall Institute of Medical Research,
Cooperative Research Centre for Cellular Growth Factors, and the
Department of Diagnostic Haematology, Royal Melbourne Hospital,
Victoria, Australia.
Submitted August IO, 1994; accepted September 20, 1994.
Supported by grants from the Victorian Health Promotion Foundation, the Anti-Cancer Council of Victoria, the National Health
and Medical Research Council, Canberra, the Cooperative Research
Centre for Cellular Growth Factors, National Institutes of Health
Grant No. CA 22556, and Chugai Pharmaceutical CO,Tokyo, Japan.
A S . was supported by fellowships from theLeukemia Society of
America and the Swiss National Science Foundation.
Address reprint requests to C.G. Begley, MD, The Walter and
Eliza HallInstituteof Medical Research, Post Ofice, TheRoyal
Melbourne Hospital, Victoria 3050, Australia.
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 1734 solely to
indicate this fact.
0 199s by The American Society of Hematology.
0006-4971/95/8S02-0012$3.00/0
379
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380
into the MPZedNeo retrovirus so that SCL expression was under
control of the myeloproliferative sarcoma virus (MPSV) long termicells were
nal repeat (LTR) promoter as previously de~cribed.’~ MI
then infected by cocultivation with an SCL-retroviral packaging cell
line.” Following 3 days of cocultivation cells were cloned in agar
by selection in G418 (400 pg/mL). On day 7, individual G418
resistant colonies were resuspended to establish four MI/SCL clonal
cell lines. MllSCL clonal cell lines were confirmed to express the
retroviral (exogenous) SCL mRNA. Three control cell lines were
obtained using the vector alone: clonal cell lines derived by G418
selection in agar (Ml/neo cell lines) were examined in addition to
parental MI cells. All cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). At
varying time points cells were prepared for morphologic examination
(using cytospin preparations stained with May-Grunwald Giemsa
and examining a minimum of 200 cells) and Northern analysis, with
medium being changed every 3 days during the culture period. Viable cell numbers were determined in triplicate by trypan blue dye
exclusion with cells counted in a hemacytometer. All cultures were
stimulated by maximal concentrations of LIF (4 ng/mL), IL-6 (32
ng/mL), or OSM (100 ng/mL) unless otherwise stated and for the
period of time indicated. Cells were removed, washed, cell counts
performed, and cells cultured in liquid cultures and in agar cultures
with or without M-CSF (500 U/mL).
Agar cultures. Cells (500/mL) were cultured with varying concentrations of purified recombinant mouse LIF, purified recombinant
murine IL-6, purified recombinant human OSM, 500 units purified
native M-CSF, or combinations of these factors as indicated. Experiments were performed in 35 mm Petri dishes using DMEM with a
final concentration of 20% pre-selected FCSand 0.3% agar in a
final volume of 1 mL. Cultures were incubated at37°Cin a fully
humidified atmosphere of 10% CO2 in air. Colonies (clones of more
than 40 cells) were scored at X 35 magnifications using a dissection
microscope after 7 days of incubation. Differentiated colonies were
identified by their characteristic dispersed morphology.5
Recloning experiments were performed by selecting consecutive
colonies that had been cultured for 7 days in maximal concentrations
of LIF. These colonies were resuspended and half the suspension
cultured with M-CSF and half with no stimulus for an additional 7
days.
Northern blotting. Poly-(A)+ mRNA was isolated from cells as
described previously by Gonda et al.3hRNA samples were size fractionated in 0.8% agarose gels, using 1 X MOPS (20 mmoVL MOPS,
1 mmol/L EDTA, S mmoVL sodium acetate), 0.22 mol/L formaldehyde. RNA wastransferred to nitrocellulose (Hybond C-extra, Amersham, Arlington Heights, IL), baked at 80°C for 2 hours, and prehybridized at 42°C for more than 1 hour in50% formamide, 4 X
Denhardt’s (0.08% Ficoll, 0.08% bovine serum albumin, 0.08%
polyvinyl-pyrolidone), 5 mmol/L EDTA, 5 X SSC, 100 pg/mL denatured salmon sperm DNA. i2P-labelled probes were derived by random priming (Bresatec Adelaide, South Australia) and hybridization
performed overnight at 42°C by adding 1 to 5 X 10‘ c p d m l to
hybridization buffer. Filters werewashedat65°Cin
0.2 X SSC,
0.1% sodium dodecyl sulfate (SDS) and exposed to film.
Flow cytometry. To assess Maclcv expression, cells were washed
in HEPES-buffered balanced salt solution (BSS) containing 5% FCS,
adjusted to 1 x lo5 cells/mL, blocked with normal mouse immunoglobulin (Ig) and then incubated for IS minutes with 1:250 dilution
of purified biotinylated anti-Macla, washed, and stained with phycoerythrin-conjugated Steptavidin for 15 minutes before washing and
resuspension in KDS containing 5% FCS and 1 pg/mL of Propidium
Iodide. All incubations were performed on ice. Cells were analyzed
on a FACScan (Becton Dickinson, Mountainview, CA) and resulting
data were analyzed usingLysys I1 software (Truefacts Software,
Seattle, WA).
TANIGAWA ET AL
Single cell autoradiography. Cells were incubated in duplicate
tubes with M-CSF (or G-CSF) radiolabeled as previously described.”~’*Cells were incubated with or without at least 100-fold
excess of unlabeled purified murine M-CSF (or G-CSF) in DulbecCO’s modified Eagle’s mediumandFCS (100 p1 final volume) at
4°C for 4 hours. Specific counts bound (total binding minus binding
in the presence of excess unlabeled M-CSF) was determined. Cytocentrifuge smears of cells labeled in the presence or absence of
excess unlabeled M-CSF (or G-CSF) were fixed in 2% glutaraldehyde in phosphate buffered saline and prepared for autoradiography
as described.” After an exposure time of 2 weeks, cell smears were
stained with 10% Giemsa in water, coverslipped and then analyzed
at X 1,000 magnification and specific grain counts determined over
labeled cells.
RESULTS
DifSerentiation induction of parental M1 cells. Figure 1
shows a northern blot analysis examining the time course of
differentiation-induction in LIF-treated parental M1 cells.
As shown previously’6 during the 4 days of treatment SCL
mRNA markedly decreased and became undetectable. The
decrease in SCL mRNA was approximately coincident with
increased levels of mRNA for several markers of macrophagedifferentiation,lysozyme,
Macla, M-CSF, and MCSF and G-CSF receptors. Interestingly, the mRNA for ID,
a negative regulator of HLH genes behaved in a reciprocal
manner to SCL mRNA, as also occurs during differentiation
induction in murine erythroleukemia cells (MEL),25 and
possibly reflecting a regulatory role for this molecule.’2 Similar
changes were observed with
a similar time course when cells
were treated with either IL-6 or OSM (data not shown).
The changes in mRNA expression were associated with
the typical morphologic features of macrophage differentiation (Fig 2). Parental M 1 cells had the appearance of undifferentiated blast cells with large nuclei, prominent nucleoli,
and scant cytoplasm (Fig 2a). Following
5 days treatment
with LIF (Fig 2c), IL-6 (Fig 2g) or OSM (data not shown)
there was clear morphologic evidence of differentiation: the
nucleus was smaller, with less prominent nucleoli and abundant
vacuolated
cytoplasm.
These changesweremore
marked after 7 days of treatment (Fig 2e). Thus, differentiation of M 1 cells in response to
LIF, IL-6, and OSM was
associated with typical morphologic changes, increased expression of mRNAs associated with
a macrophage phenotype,and a late ( 3 to 4 days)decreaseinlevelsof
SCL
mRNA.
Behavior of M I and MUSCL cells in response to LIF,
OSM, and IL-6. To explorethepossibleroleofSCLin
macrophage differentiation of M 1 cells, an SCL retrovirus
was used to infect parental M1 cells. Four clonal cell lines
in which production of
SCL mRNA and protein was enforced
(M USCL cell lines), and four controlcell lines infected with
a retrovirus containing only the neomycin resistance gene
( M l h e o cell lines) were established. These cell lines were
analyzed for their response to differentiation induction by
LIF, IL-6, and OSM.
Figure 2 shows the morphology of Ml/SCL cells compared with parental cells treated with
LIF or IL-6. In contrast
to parental cells or control M l h e o cell lines, differentiation
was perturbed in Ml/SCL cells treated with LIF (Fig 2) or
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381
FERENTIATION SCL AND MACROPHAGE
CIF
None
Probe
24
20s18s -
n8&8
Lysozyme
Macla
M-CSF
M - CSF
Receptor
Fig l. Northern blot analysis of poly(A)+ mRNA
extracted from M1 cells after 0, '/,, l/*, 1,2, or 4 days
(as indicated). Cells were treated with
LIF (leftpanel)
or untreated. The Dosition of 28s and 18s ribosomal
RNA is shown. The probes used are indicated on the
right.
OSM (data not shown). After 5 days treatment the MI/SCL
cells showed less abundant cytoplasm, fewer vacuoles and
larger, more immature nuclei (Fig 2d) compared with parental cells (Fig 2c). This difference was also evident after 7
days treatment with LIF or OSM (Fig 2f). Similar behavior
was observed with all four MI/SCL clonal cell lines, while
the response of all Ml/neo cell lines was identical to parental
MI cells.
Surprisingly however, following treatment with IL-6, the
Ml/SCL cells were morphologically indistinguishable from
parental MI cells (or Ml/neo cells). As shown in Fig 2h, the
Ml/SCL cells showed abundant cytoplasm with prominent
vacuolation and nuclear maturation after 5 days treatment
with IL-6. This phenotype was observed with all four MI/
SCL cell lines.
Similar results were observed when cells were examined
for expression of Macla antigen following growth factor
G-CSF
Receptor
o r m o w
00
GAPOH
treatment (Fig 3). As expected, after 5 days treatment with
growth factor, parental MI cells showed increased surface
expression of Macla compared with untreated cells (Fig 3A
and B). A similar increase in Macla expression was observed with M I/SCL cells treated with 1L-6 (Fig 3C and D).
However, consistent with the morphologic data, when M11
SCL cells were treated with LIF or OSM, the increased
Macla expression was approximately 100-fold less than that
observed with parental andM I/neo cells or IL-6-treated M l /
SCL cells. Thus, enforced SCL expression resulted in disturbed differentiation following treatment with LIF or OSM,
but not IL-6.
Northernblot analysis also showed evidence of a perturbed response to LIF (Fig 4) or OSM (data not shown) in
the Ml/SCL cells. Early events were unaltered, but the increase in lysozyme mRNAwas delayed. Consistent with
the results presented above, northern analysis showedno
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TANIGAWA ET AL
382
M1
MVSCL
Saline
(Sd)
"
C
LIF
(Sd)
LIF
(7d)
Y
"
IC-6
(Sd)
difference between parental MI cells and MI/SCL cells
treated with IL-6 (data not shown). Unexpectedly, enforced
SCL had no effect on LIF or OSM-induced expression of
M-CSF receptor or G-CSF receptor mRNA (see below).
Clonal ana1.ysis of M I and Ml/SCL cells. When analyzed in clonal agar cultures, the MI/SCL cells also displayed an altered dose-response relationship to LIF and
OSM, but not IL-6. With parental M1 and control Ml/neo
cell lines, all three growth factors resulted in 1 0 0 % differentiated colonies. This was also true for all MI/SCL clonal
cell lines treated with IL-6. However, in cultures of MI/SCL
cell lines treatedwith LIF or OSM, only a proportion of
colonies showed a differentiated phenotype (Fig 5). To examine this further, LIF-treated differentiated colonies and
LIF-treated undifferentiated colonies were isolated, expanded, and clonal sublines of MI/SCL cells established.
,
Fig 2. Morphology of parental M1 cells and a typical clonal cell line carrying an SCL retrovirus (M11
SCL cells). Panels a and b show untreated cells held
for 5 days in liquid culturescompared with cells stirnulated with LIF for 5 days (panels c and d) or 7 days
(panels e and f ) or 5 days with IL-6(panels g and h).
Note similar morphology ofM1 and MllSCL cells in
panels a,b and g,h versus disturbed differentiation
of MllSCL cells in response t o LIF (panels d and 1).
This experiment was typical of results with the 3
M l l n e o and the4 MllSCL cell lines.
The response of these sublines to LIF was then reexamined.
Inall cases these secondary sublines displayed a similar
reduced response to LIF thatwas comparable to that observed with the initial four MI/SCL clonal cell lines (Fig
SD).Thus, it was not possible to select an MI/SCL subclone
that could be completely suppressed by LIF. Similar results
were also obtained in direct colony recloning assays where
neither LIF nor OSM could completely extinguish MI/SCL
clonogenic cells (see below), although all three growth factors could completely suppress parental and control Ml/neo
cells."'
Experiments were also performed to examine the response
of MI/SCL cell lines to combinations of growth factors.
Table 1 shows results of experiments comparing equivalent
biologic concentrations of LIF and IL-6 (4 ng/mL and 32
ng/mL, respectively, a fourfold maximal concentration, see
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SCL AND MACROPHAGEDIFFERENTIATION
I
383
M
c
of colonies (maximum of 45%) displayed a differentiated
phenotype (data not shown).
Thus, in summary the enforced expression of SCL in MI/
SCL cells resulted in perturbation of several aspects of the
macrophage differentiation program as evidenced by cellular
morphology, expression of lysozyme and MacIa, and functional ability to migrate through agar. Associated with this
inability to complete a terminal differentiation program in
response to LIF and OSM (but not IL-6), a proportion of
MI/SCL cells maintained their clonogenicity and their proliferative ability was not suppressed.
M-CSFresponsefolk[~wingLIF/IL-UOSMtreatment. Following differentiation-induction of MI cells by LIF (Figs 1
and 4), IL-6, or OSM (data not shown) there was increased
expression of mRNAs for M-CSF receptor and G-CSF receptor. An equivalent increase was also seen in MI/SCL cells
regardless of the growth factor under study (Fig 4). Therefore, experiments wereperformedto further evaluate this
apparent dissociation between the aspects of monocytic dif-
B
D
I F
L1F
M1
0
01
1
l
MVSCL
0 2 4 6
H
p--
." ' "
M t T
28s 18s100
101
102
l# 104 100
101
102
103
0 2 4 6
-
r
SCL
104
c-myb
LOG FLUORESCENCE
Fig 3. Flow cytometricanalysis of M a c l n antigen expression. Panels on the left(A, C, E, and GI show Maclaexpression (solid curve)
for control, untreated cells, and panels on right(B, D, F, and H) show
M a c l a expression following 5 days treatment with growth factor.
The black line indicates background staining determined using an
isotype-matched control antibody. Panels A and B show increased
M a c l a expression on parental M1
cells following treatment with
LIF.
Increased expression of Macla on Ml/SCLcells is shown following
treatment with IL-6 (panels C and D) compared with Ml/SCL cells
treated with LIF (Panels E and F) or OSM (Panels G and H). Fluorescence is shown on a logarithmic state.
Fig 5) and experiments using a SO-fold excess of LIF over
IL-6 (200 ng/mL versus 32 ng/mL, respectively). When used
with LIF, IL-6 induced 100% differentiation in clonogenic
MUSCL cells. Similarly, in liquid cultures the combination
of IL-6 and LIF resulted in 100% differentiated cells after
7 days in culture (data not shown). This compared with a
maximum of 40% of cells showing some differentiated features in liquid cultures stimulated by LIF alone. The effect
of combining LIF and OSM was also examined (data not
shown). M I/SCL cells from the four clonal cell lines, when
treated with LIF and OSM together, behaved as though stimulated with only a single growth factor and only a proportion
-9
Lysozyme
M-CSF
Receptor
G -CSF
Receptor
GAPDH
Fig 4. Northern blot analysis of poly(A)+ mRNA extracted from
M1 and MllSCL cells after treatment with LIF for 0, 2, 4, or 6 days
RNA is shown.
(as indicated). The position of28s and 18s ribosomal
The probes used are indicated on the right. Similar results
were obtained when cells were treated with OSM (not shown).
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384
TANIGAWA ET AL
1
B
A
OSM (ng/ml)
LIF (ng/ml)
Fig 5. Dose-response relationship for cells treated with LIF (panel
A), IL-6 (panel B) or OSM (panel C) and analyzed in clonal cultures.
Results are shown for control M1 cell lines (open symbols; parental
M 1 cells [Of) and for several MlISCL clonal cell lines (closed symbols). Panel D shows responsiveness of four sublines
generated from
Two sublines were obtained from
one clonal Ml/SCL cell line
two differentiated colonies in a LIF-stimulated culture ( + 1 and two
sublines from two undifterentiated colonies in a LIF-stimulated culture (A).Results are means of triplicate cultures.
cell populations (Fig 6A and B). Following treatment of cells
with LIF, therewas a marked increase in binding with a
minimum of 25% to 35% of cells showing specific grains.
A subpopulation of these cells were again prominent because
of their intense labeling (Fig 6C and D). There was no specific labeling of cells with G-CSF before LIF treatment and,
after LIF treatment there was an even, low level distribution
of grains throughout the cell populations. Again there was
no difference between parental MI, Ml/neo, and MI/SCL
cells (data not shown).
The behavior of cells in M-CSF and G-CSF stimulated
cultures was also examined. As reported previously for the
parental M1
there
was
no
detectable effect Mof
CSF (or G-CSF) alone on untreated M1 cells or on the M1/
SCL cells (data not shown) either in terms of stimulated
proliferation or differentiation-induction. Parental M l , M l /
neo,and Ml/SCL cells were, therefore, treated in liquid
cultures with LIF. OSM, or IL-6 for 4 days to induce expression of M-CSF and G-CSF receptors. Cells were then washed
and placed in liquid cultures either with or without M-CSF
Table 1. Effect of LIF and IL-6 Together on M 1 and Ml/SCL Cells
Stimulus
Cells
Parental
M1
(m).
+
Ml/neo
ferentiation outlined above and M-CSF receptor/G-CSF receptor status in LIF-treated Ml/SCL cells.
Binding studies were performed with radio-iodinated MCSF and G-CSF to confirm that the LIF-induced increase
in M-CSF receptor/G-CSF receptor mRNA expression was
associated with increased protein production. After treatment
with LIF, all cell populations displayed increased and equivalent levels of M-CSF binding (eg, 1,848 specific counts
bound (scb) per 10' untreated parental M1 cells versus 5,509
scb per lo5LIF-treated parental M1 cells: 1,928 scb per 10"
untreated Ml/SCL cells versus 4,570 scb per 10' LIF-treated
Ml/SCL cells.) Similarly, after LIF treatment, specific GCSF binding was detected on cells from parental M1, M I /
neo and MllSCL cell lines, although the degree of binding
was less than that observed with M-CSF (eg, no detectable
scb before LIF treatment versus 490 scb per lo5 LIF-treated
Ml/neo cells: 615 scb per lo5 LIF-treated Ml/SCL cells).
Single cell autoradiography was also used to examine specific binding of M-CSF and G-CSF and, again there was no
detectable difference between control M1 cells and Ml/SCL
cells. Consistent with the low level binding of M-CSF observed with untreated control M l and Ml/SCL cell populations, M-CSF binding was detected by autoradiography. This
binding was due to a subpopulation of M1 cells that labeled
intensely with M-CSF and represented between 1% and 2%
(minimum 900 cells examined) of control M1 or Ml/SCL
Saline
LIF (4 ng/mL)
LIF (200ng/mL)
IL-6 (32 ng/mL)
IL-6 (32 ng/mL)
LIF (4 ng/mL)
IL-6 (32 ng/mL)
+ LIF (200 ng/mL)
Saline
LIF (4 ng/mL)
LIF (200ng/mL)
IL-6 (32 ng/mL)
IL-6 (32 ng/mL)
+ LIF (4 ng/mL)
IL-6 (32 ng/mL)
+ LIF (200ng/mL)
Saline
LIF (4 ng/mL)
LIF (200ng/mL)
IL-6 (32 ng/mL)
IL-6 (32 ng/mL)
LIF (4 ng/mL)
IL-6 (32 ng/mL)
LlF (200 ng/rnL)
Saline
LIF (4 ng/mL)
LIF (200ng/mL)
IL-6 (32 ng/mL)
IL-6 (32 ng/mL)
LIF 14 ng/mL)
IL-6 (32 ng/mL)
+ LIF (200ng/mL)
Ml/SCL (4)
Ml/SCL (8)
Colony
Number
Differentiated
Clones (%l
368 i 10
182 ? 8
176 f 5
51 2 3
0 .t 0
100 2 0
100 i 0
100 -+ 0
100 2 0
6 5 2
100
7
380
222
215
24
2 2
t 14
f6
i5
-t 3
020
100 i 0
100 f 0
100 +- 0
100 2 0
3 2 1
100 t 0
3+-1
420 ? 12
369 2 17
360 f 12
24 f 3
+
16 i 2
+
15 +- 3
392 2 13
312 -+ 6
318 f 8
25 -t 4
+
z0
020
40 f 5
38 f 2
100 t 0
100 -+ 0
100 f 0
Of0
48 i 5
42 ? 7
100 -t 0
100 2 0
15 i 3
100 i 0
13 t 2
Cultures contained500 parental M1, Ml/neo, or Ml/SCLcells (clonal
cell lines 4 and 8) plus saline, purified recombinant LIF or purified
recombinant 11-6 as indicated. Colonies were counted and typed after
7 days incubation. Results are meanf standard deviation from duplicate cultures in two experiments.
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SCL AND MACROPHAGEDIFFERENTIATION
385
-
U
'*
*
I
i
Fig 6. Single cell autoradiography using radiolabeled M-CSF and examining parentalM1 cells (panels A and C) and Ml/SCL cells (panels
B and D). Cells were either unstimulated (panels A and B) or stimulated with LIF (panels C and D). Note rare, intensely labeling cells in the
unstimulated populations. Following treatment with LIF (3 days) there was increased labeling of both parental M1 and MllSCL cells, but in
the absence of morphologic changes of terminal differentiation of Ml/SCL cells. Note smaller cell size, less abundant cytoplasm in panel D
compared with panel C.
(or G-CSF). Figure 7 shows cell growth curves for such
an experiment. Consistent with the ability of LIF to totally
suppress the proliferative ability of parental MI (and M1/
neo) cells, cell numbers continued to decline following withdrawal of LIF. Furthermore, those viable cells that did persist
showed a terminally differentiated phenotype. In contrast,
M-CSF stimulated the proliferation of parental M 1 cells that
hadbeenpretreatedwith
LIF, TL-6. or OSM. M-CSF had
no such growth-stimulatory effect on MI cells that had not
been pretreated with the cytokines. The altered behavior of
LTF (or 0SM)-treated Ml/SCL cells compared withLIFtreated parental cells was again evidenced following LIF
withdrawal: MI/SCL cells continued to proliferate. However, proliferation was still dramatically enhanced when cells
were treatedwith M-CSF andwas indistinguishable from
the behavior of pretreated parental M I cells (Fig 7). In exper-
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386
TANIGAWA ET AL
M1
onies were selected and resuspended. Half the colony was
replated in a secondary culture with no stimulus and half the
colony replated in a secondary culture stimulated by M-CSF
(Fig 8). Undifferentiated colonies gave rise to an increased
number of secondary colonies when stimulated by M-CSF.
This suggested that although these cells had failed to respond
to LIF in terms of their ability to migrate through agar, they
had responded to LIF in terms of the acquired ability to
respond to M-CSF. As expected, differentiated, LIF-responsive Ml/SCL colonies also showed increased numbers of
secondary colonies when stimulated by M-CSF. As reported
previously M-CSF was also able to rescue LIF-treated parental M1
M1 ISCL
l00
1
osayne
- OMCSF
-
50
-
50
l
l
1
2
l
3
DISCUSSION
0
1
2
3
Days in culture
Fig 7. Proliferation of M1 and Ml/SCL cells stimulatedby M-CSF
after 4 days preincubation with LIF. After stimulation with LIF, cells
were washed and placedin liquid cultures stimulated by M-CSF or
with n o stimulus. Results are mean
5 standard deviationof triplicate
cell counts performed after
0 to 3 days in M-CSF. Similar results were
obtained with all Ml/SCL clonal celllines.
iments with G-CSF-stimulated cells following LIF, IL-6, or
OSM pretreatment, G-CSF did not stimulate proliferation
but appeared to enhance cell survival, and its effect on the
parental M1 and MI/SCL cells was the same (data not
shown).
Similar results were observed in clonal cultures. Cells
were pretreated with LIF, IL-6, or OSM in liquid cultures
for 4 days, washed, and cultured in agar for 7 days. Typical
results are shown in Table 2. Pretreatment of parental M1
(and Mlheo) cells with LIF or IL-6 and continued culture
in LIF or IL-6 resulted in marked suppression of clonogenic
cells from approximately 80% clonogenic cells (see Table
1) to0.6% (Table 2). Moreover, thefew clones thatdid
proliferate displayed a differentiated phenotype (data not
shown). Stimulation by M-CSF in agar cultures was able to
rescue parental M1 cells from LIF or IL-6 induced extinction, and there was a significant increase in the number of
clonogenic cells. The "CL
cells were essentially extin- (4)
guished by IL-6, butnotby LIF. After either LIF or IL-6
treatment, there was enhanced colony growth in cultures of
Ml/SCL cells stimulated by M-CSF (Table 2). The fre- (8)
quency of clonogenic cells was approximately 50%, suggesting that at least half the cells within these populations
were able to respond to M-CSF.
The dissociation between SCLs ability to interfere with
some aspects of the macrophage differentiation
program (eg,
Saline
IL-6
morphology, migration, suppressed proliferation) butnot
other aspects (eg, M-CSF response) was unexpected. Therefore, clone transfer experiments were performed to determine whether this dissociation applied at a clonal level. MI/
SCL cells were stimulated by LIF in agar cultures for 7 days.
At this time it was easy to distinguish colonies that displayed
a differentiated phenotype from those that did not. Consecutive undifferentiated, and apparently LIF nonresponsive col-
Murine myeloid leukemia cell lines such as WEHI-3B
andM1have
long served as models for the physiologic
induction of terminal differentiation to macrophages and
g r a n ~ l o c y t e s ' . ~and
. ~ ~have
- ~ ~ helped in the identification of
several natural inducers of differentiation including G-CSF,
LIF (D-factor), IL-6 (MG1-2), and OSM.2.3.4.4'While initially these differentiation inducers appeared to have little in
common, recent data have suggested that they are evolutionarily related, probablyretain a very similar three-dimensional structure (long chain four-a-helical bundle), and utilize cellular receptors that are all members of a structurally
conserved hematopoietin
Even more interesting,
at least three of these differentiation inducers (LIF, IL-6,
and OSM) share a common receptor subunit (gp130) that is
essential for biologic signalling.'"' These observations have
Table 2. Effect of M-CSF o n Colony Growth of LIF-treated
or IL-6 treated M1 Cells
Cells
Stimulus
(liquid culture)
Parental M 1
LIF
Ml/neo
LIF
Ml/SCL
LIF
Ml/SCL
LIF
Parental M 1
IL-6
Stimulus
(Agar culture)
Saline
M-CSF
LIF
Saline
M-CSF
LIF
Saline
M-CSF
LIF
Saline
M-CSF
108 LIF
Saline
M-CSF
IL-6
Ml/SCL (8)
M-CSF
IL-6
Colony
Number
23 f 2
112 2 8
3 2 1
19 2 2
159 f 16
2 2 1
144 f 16
358 2 19
92 2 4
125 f 17
249 2 26
f8
11 5 2
58 f 6
4 5 1
9 2 1
38 5 2
3 5 1
Cells (parental M1, Ml/neo, Ml/SCL clonal cell lines 4 and 8) were
Stimulated with LIF (4 ng/mL) or IL-6 (32 ng/mL) for 4 days in liquid
cultures. Cells were washed, counted, and 500 cells examinedin agar
cultures stimulated with M-CSF (500 units). LIF, IL-6, or saline as indicated. Colonies were counted after 7 days. Results are mean 5 standard deviation from duplicate culturesin two experiments.
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
387
SCL AND MACROPHAGEDIFFERENTIATION
receptor, morphologic differentiation to macrophages, and
suppression of proliferative capacity. Because SCL has been
implicated in both cell proliferation and differentia ti or^?^^^',^^
we asked whether enforced expression of SCL would prevent
470
or perturb the program of differentiation in M1 cells induced
with LIF, IL-6, or OSM. WhenSCL expression was enforced
by infection of M1 cells with anMPZen retrovirus containing
400
SCL cDNA, there were no observed changes in the behavior
of untreated cells. However, enforced SCL expression resulted in a perturbed pattern of differentiation induction by
LIF and OSM. The suppression of myb and later induction
of M-CSF receptor and G-CSF receptor mRNA and protein
were essentially the same for M1 and Ml/SCL cells induced
with LIF or OSM, but the induction of lysozyme, Macla,
300
morphologic differentiation, and suppression of proliferation
were all significantly reduced in "CL
cells and were
confirmed at the clonal level (Fig 8). These results indicated
that distinct signaling pathways were responsible for different elements of the normal differentiation induction pathway
in M1 cells and that SCL could interfere with some elements,
but not others. This was not just a matter of SCL interfering
200
with downstream differentiation events but not earlier
events, because the induction of lysozyme and Macla (affected by SCL) and the induction of M-CSF and G-CSF
receptors (not affected by SCL) were all relatively late
events.
Also of interest, enforced SCL expression disturbed differ100
entiation induction by LIF and OSM, but not by IL-6. This
was surprising because all three differentiation inducers
share a common receptor signaling subunit (gp130). The
initial signaling events in M1 cells appeared to be identical
for LIF and IL-6'2"4and OSM (present work), and enforced
expression of myc or myb in MI cells did not result in a
I
dissociation between LIF/OSM and IL-6 response^!^.^^ The
MCSF
M-CSF =
differential response to LIF/OSM versus E - 6 in Ml/SCL
cells wasnot due to altered numbers or affinities of cell
Fig 8. M-CSF response of cells from colonies after 7 days culture
surface receptors for these cytokines16and must presumably
in UF. Consecutive UF-induced differentiated or undtferentiated col- reflect differences in the intracellular cascades triggered by
onies from one clonal
MllSCL cell line wereselected, washed, resuspended, and half the colony recultured in M-CSF and half the colonythese cytokines.
reculturedwith no stimulus. The number of secondary colonies were The current view of cellular receptors for IL-6, LIF and
enumerated 1 week later. Results for four LIF-induced dtferentiated OSM might provide a mechanism for the selective effect of
colonies fromthe parental M1 cell line are also shown
(0).Note the
SCL on LIF and OSM signaling. IL-6 receptors consist of
(UF-induced)abilii of M-CSF to stimulate secondary colony formaa unique a-chain and gp130 but homodimerization of gp130
tion from coloniesthat were (apparently) LIF-unresponsive.
is thought to be essential for ~ignaling.'.~'.~~
The a-chain of
the LIF receptor (LIFR) is unlike the IL-6 receptor a-chain,
butmore like gp130 and the active receptor complex is
provided a unifying concept to explain the biologic redunthought to be a LIFlUgpl30 heterodimer. OSM binds to
dancy of these cytokines, but have failed to clarify if and
gp130, but it, too, utilizes an identical LIFlUgpl30 heterohow the individual cytokines exert unique biologic actions
dimer for cellular ~ i g n a l i n g . ~ We
. ~ . ~propose
~ . ~ ~ that gp130/
other than what might be expected by unique patterns of
LIFR heterodimers use an SCL-inhibitable signaling pathreceptor expression on different cells. In the present study,
way to induce morphologic differentiation of M1 cells, the
we have addressed this question by examining the effect
of enforced expression of the SCL transcription factor on
capacity to migrate in agar and proliferative suppression, but
differentiation-induction in murine M1 cells, which simultathat gp130 homodimers can utilize an alternate pathway. The
neously display receptors for LIF, IL-6, and OSM.
assembly of different pairs of JAK-related kinases by the
The effects of treatment of parental M1 cells with LW,
two different types of receptor dimer might provide for the
IL-6, or OSM were essentially identical, with early suppresactivation of different intracellular pathways.13.14
Both recepsion of endogenous myb and later suppression of endogenous
tor complexes utilize alternate pathways (not SCL-inhibitSCL expression, followed by induction of lysozyme, the
able) to induce M-CSF receptor and G-CSF receptor exprescell surface markers Macla, M-CSF receptor and G-CSF
sion (Fig 9).
1
+
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
388
TANIGAWA ET AL
LFlosM
IL-6
0
Fig 9. Model of LIF/OSM and IL-6 signaling in M 1 cells as determined by enforced expression of SCL. The decrease in myb expression occurred early (possibly via several
different pathways) and may
or may not be directly involved in the later decrease in expression
of SCL. However in response to LIF and OSM, enforced expression
of SCL perturbed cellular morphology, lysozyme,and M a c h expression, ability to migrate, and clonogenicity. The increased expression
of M-CSF and G-CSF receptors occurred independently of SCL. Presumably similar intracellular pathways exist in response to IL-6 signaling through a gp130 homodimer. In addition a pathway(s) that
was SCL-independent resulted in a differentiated phenotype.
Although SCL expression significantly reduced the induction of some aspects of phenotypic differentiation by LIF
and OSM, it did not completely abrogate it. In fact, a proportion of Ml/SCL colonies (20% to 40%) displayed apparently
typical differentiation-induction, albeit requiring higher concentrations of LIF or OSM (Fig 5). This did not represent
clonal heterogeneity of M1/SCL cells because sublines established either from differentiated or undifferentiated colonies continued to express SCL and regenerated the same 20%
to 40% responsiveness to LIF. This behavior must reflect a
stochastic event and, given the model of Fig 9, we suggest
that, at a low frequency, LIF or OSM might recruit gp130
dimers rather than monomers into their receptor complexes
and thereby gain IL-6-like signaling capacity. This would be
consistent with the increased dose-response in Fig 5 because
higher levels of receptor occupancy would be required to
achieve these rare events.
The (SCL-independent) induced expression of M-CSF receptors on M1 cells and generation of M-CSF responsiveness
is a well documented component of normal macrophage differentiation” and of differentiated M1 cells.56Surprisingly,
we found that a very small subpopulation (1% to 2%) of
untreated M1 cells expressed high levels of M-CSF receptors
(Fig 6) and because some of the cells labeled by autoradiography appeared relatively mature, this may reflect a low level
of spontaneous differentiation. Nevertheless, this low level
of expression did not result in any detectable effect of MCSF on M1 cell growth or differentiati~n.’.~’ LIFor IL-6induced differentiation of M1 cells led to a marked increase
in the proportion of cells expressing high levels of M-CSF
receptor (25% to 35%) and these cells were now able to
survive and proliferate in response to M-CSF. Despite the
lack of complete differentiation of Ml/SCL cells in LIF.
they responded similarly with respect to M-CSF receptor
expression and M-CSF proliferative responsiveness.
The transcription factor SCL has been implicated in both
differentiation and proliferation events.’”,48SCL hasbeen
suggested to play a positive role as a regulator of erythroid
differentiation and may also be involved in neural differentiati~n.~’.~’
The level of SCLmRNA has been reported to
decrease during myeloiddifferentiation16,”andthe results
reported here suggest thatthe decrease in SCL mRNAis
required for macrophage differentiation to proceed normally.
Whether this is a direct action of SCL protein itself, or a
consequence of the overexpressed SCL sequestering another
protein that is itself required for differentiation to proceed
normally is unknown. It is also possible that SCL exerted
an apparent effect on differentiation by preventing growtharrest (which might normally trigger an intrinsic program
of differentiation). SCL is likely to play a role in cellular
proliferation. Its frequent involvement in human T-cell leukemia,2’,22,23
its activation byan intracisternal-A particle
insertion in WEH1-3BD-~ells,~*
and its role to enhance clonogenicity and tumorigenicity in a murine T-cell line,’4 all
suggest SCL may provide a proliferative advantage for cells.
More direct evidence comes from experiments in which an
antisense strategy interfered with cellgrowth.48In the experiments presented here, enforced SCL expression did not provide a detectable growth advantage as such, but did allow
cells to escape growth factor-induced suppression of proliferation.
This system may provide a useful model to analyze molecules involved in growth factor-receptor signaling and macrophage differentiation and could also be used to further
examine the effects of SCL and related HLH transcription
factors.
ACKNOWLEDGMENTS
We are indebted to Doug Hilton for helpful discussions. We wish
to thank Cathy Owczarek and Meredith Layton for the recombinant
human OSM, and Richard Simpson for recombinant mouse IL-6.
We thank Paula Gason and Caroyln Farley for expert technical assistance.
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1995 85: 379-390
Differential regulation of macrophage differentiation in response to
leukemia inhibitory factor/oncostatin-M/interleukin-6: the effect of
enforced expression of the SCL transcription factor
T Tanigawa, N Nicola, GA McArthur, A Strasser and CG Begley
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