Regulation of Human Monocyte DNA Synthesis by Colony-Stimulating Factors,
Cyto kines, and Cyclic Adenosine Monophosphate
By Daisy L. Cheung and John A. Hamilton
It is reported in this study that a subpopulation of highly
purified human peripheral blood human monocytes can
proliferate in response to colony-stimulatingfactor-1 (CSFl), granulocyte-macrophage colony-stimulatingfactor (GMCSF), and interleukin-3 (IL-3). Both GM-CSF and IL-3 synergized with CSF-1 for the induction of DNA synthesis. Given
the DNA synthesis levels attained, we were able to test the
effects of certain cytokines and cyclic adenosine monophosphate (CAMP)-elevating agents, which have been shown to
modulate in vitro human myelopoiesis and murine macrophage proliferation.The cytokines, interferon-y(IFN-y), inter-
leukin-4 (IL-4). and tumor necrosis factor-alpha (TNF-a), as
well as CAMP-elevating agents, 8-bromoadenosine 3’:5’cyclic monophosphate (BBrcAMP), cholera toxin (CT), and
prostaglandin E, (PGE,), suppressed the monocyte DNA
synthesis due to CSF-1. These results parallel those reported
with human bone marrow progenitors, as well as murine
macrophage populations. The cycling human monocyte population could provide a model cell type to understand the
molecular events controllinghuman myelopoiesis.
0 1992 by TheAmerican Society of Hematology.
A
Most of the current knowledge of the signals involved in
the control of the proliferation of macrophages and their
progenitors by CSFs has been obtained from the findings of
experiments in which CSF-l-dependent immortalized murine cell lines and murine BMM have been used as cell
models.” Among the important biochemical signals implicated in the negative control of CSF-mediated proliferation
are those that result from elevations in cyclic adenosine
monophosphate (CAMP) levels, for example, in the inhibition of BMM DNA synthesis stimulated by CSF-1, granulocyte-macrophage CSF (GM-CSF), or interleukin-3” (IL-3)
(also known as multi-CSF), and that of BACl.2F5 cells,
stimulated by CSF-1.” Indirect evidence of the negative
regulatory role of CAMP in human myelopoiesis comes
from the finding that prostaglandin E, (PGE,), which
elevates intracellular CAMPconcentrations in human monoc y t e ~ and
’ ~ murine BMM,” can inhibit human myeloid cell
colony formation mediated by human monocyte-derived
CSF(s).I4Recently, several cytokines have been recognized
to be involved in the regulation of hematopoiesis, either as
cofactors with CSF or as colony-stimulating activities themselves. Interleukin-1 (IL-l), previously known as hemopoietin-1, promotes the survival of human myeloid progenitor^'^
and of murine myeloid progenitors.16 Interleukin-4 (IL-4)
inhibits macrophage colony formation in human bone
marrow c~ltures.’~
Other cytokines that have been involved
in the regulation of hematopoiesis include interferon-y
(IFN-y), tumor necrosis factor (TNF), and interleukin6.18-21
The present studies demonstrate that a subpopulation(s)
of human peripheral blood monocytes can undergo DNA
synthesis in response to CSF-1. With the level of DNA
synthesis observed, we were able to explore the actions of a
series of agents that are known to modulate human myelopoiesis in vitro.
PART FROM ACTING on primitive hematopoietic
progenitor cells, colony-stimulating factor-1 (CSF-1)
or macrophage CSF (M-CSF) maintains the survival and
promotes the proliferation and differentiation of mature
mononuclear phagocytes.’ Different populations of murine
mononuclear phagocytes, such as peritoneal exudate macrophages (PEM), peripheral blood monocytes, and bone
marrow cells, respond to human urinary CSF-1 for macrophage colony formation? While human CSF-1 is a potent
stimulator of murine macrophage colony formation, it
initiates a relatively weaker proliferative response in human
target cells, for instance, macrophage colony formation by
human bone marrow progenitor cells in
While
greater than 90%of murine bone marrow-derived macrophages (BMM) proliferate in response to human recombinant
CSF-1,6only up to 5% of human peripheral blood monocytes have been reported to undergo DNA synthesis on
exposure to CSF-1.7The lack of a substantial proliferative
response of these cells of the human mononuclear phagocyte lineage has presumably deterred active investigation of
the underlying molecular mechanisms involved in controlling their proliferation. Nevertheless, it was described that
CSF-1 stimulation of human monocyte DNA synthesis is
totally suppressed by pertussis toxin (PT), suggesting that
CSF-1 elicits mitogenic signals via a PT-sensitive guanine
nucleotide-binding protein’ (G-protein). In a subsequent
study, CSF-1 was shown to activate protein kinase C in
human monocytes.’ This activation of protein kinase C was
associated with increased phosphatidylcholine turnover
and 1,2-diacyglycerol (DAG) production.
From the University of Melboume, Deparlment of Medicine, Royal
Melboume Hospital, Parkville, Australia.
Submitted August 5, 1991; accepted December 16, 1991.
Supported by a Program Grant from the National Health and
Medical Research Council ofAustralia.
Address reprint requests to John A . Hamilton, PhD, DSc, University
of Melboume, Department of Medicine, Royal Melboume Hospital,
Parkville, 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 I734 solely to
indicate thk fact.
Q 1992 by The American Society of Hematology.
0006-4971I92 I 7908-OO02$3.00/0
1972
MATERIALS AND METHODS
Monocyte isolation and culture. Bu@ coats from normal donors
for isolating peripheral blood monocytes were kindly provided by
the Melbourne Red Cross Blood Bank. Monocytes were isolated by
density gradient centrifugation followed by countercurrent centrifugal elutriation (Beckman JE-6B Elutriation System, Palo Alto,
CA) as described by Hart et al.” Monocyte-enriched fractions
( t 95% purity) were resuspended in Alpha-Modified Eagle’s
Medium (aMEM, Flow Laboratories, McLean, VA), supplemented with 20 mmol/L 3-(N-morpholino) propane-sulfonic acid
Blood, Vol79, No 8 (April 15), 1992: pp 1972-1981
HUMAN MONOCYTE DNA SYNTHESIS
(Sigma, St Louis, MO), 13.3 mmol/L NaHCO,, 5 X lo-' mol/L
2-mercaptoethanol, and 70 pg/mL neomycin sulfate (complete
aMEM; osmolality, 290 mmol/kg). The purity of the elutriated
fractions was always confirmed by May-Griinwald Giemsa staining
of cytocentrifuged preparations and by nonspecific esterase staining.22The monocyte-rich fractions were then adhered to tissue
culture surfaces. In some experiments, they were plated in 24-well
multidishes (16-mm well diameter; Intermed, Nunc, Denmark) at a
density of 0.5 to 0.8 x 10' cells/well in 1mL complete aMEM. The
cultures were incubated for 90 minutes at 37°C; nonadherent cells
were removed by multiple washes with warm medium. For some
other later experiments, the highly purified elutriated monocytes
were seeded in 96-well microwell plates (5-mm well diameter;
Nunc, Roskilde, Denmark) at a density of 0.6 x 105 cellslwell in 0.2
mL complete aMEM supplemented with 1% platelet-poor plasmaderived human serum (HS) (see below) and cultured overnight at
37°C. Nonadherent cells were removed by multiple washes with
warm medium. The resulting adherent cell monolayers in 24-well
multidishes and 96-well microwell plates were cultured in 1mL and
0.2 mL complete aMEM, respectively. Since bacterial endotoxin
(lipopolysaccharide [LPS]) can inhibit monocyte DNA synthesis
(data not shown), all cultures contained 3% HS and 1 pg/mL
polymyxin B sulfate (Sigma), an agent that inhibits LPS binding to
cell membranesu Unless otherwise specified, appropriate stimuli
at the concentrations specified were added at the initiation of
cultures and incubated for up to 13 days. Control cells received an
appropriate aliquot of complete aMEM supplemented with 3%
HS. All test variables were examined in triplicate cultures.
When monocytes were cultured in microtiter plates instead of
24-well plates and CSF-1 was added after a overnight adherence
period instead of the 90-minute adherence period, there was little,
if any, difference in the pattern of response (data not shown). The
advantage of using microtiter plates is that less cells were required
for each experiment. It was because of this reason that the latter
method was adopted as the standard procedure, unless otherwise
indicated.
Measurement of DNA synthesis. As described previously," the
level of DNA synthesis was monitored by measuring the incorporation of 3H-thymidine ('H-TdR) into stimulated monocytes that
were pulsed with 'H-TdR (2.5 pCi/mL) during the last 24 hours of
incubation. For cells cultured in 24-well plates, the labeling period
was ended by removing culture medium and cells solubilized in 0.5
mL of NaOH (0.2 mol/L). To 200 pL of the solubilized cell
fraction, an equal volume of 20% trichloroacetic acid (TCA) was
added and the mixture left overnight at 4°C. The insoluble TCA
material was collected onto glass microfiber filters (Whatman
GF/C, Maidstone, England) using a Millipore vacuum harvester.
For cells cultured in microtiter plates, cellular macromolecules
labeled with 'H-TdR were collected onto glass microfiber filters
(Inotech, Lansing, MI) by means of an Inotech cell harvest system
in which cells were subjected to repeated flushing with deionized
water and vacuum suction. Radioactivity associated with the filters
was determined by p-scintillation counting.
Measurement of cell number. At the time points indicated, the
cultures were washed gently with calcium- and magnesium-free
phosphate-buffered saline, pH 7.4 (PD). The adherent cells were
lysed with zapoglobin (10% vol/vol) (Coulter Electronics,
Brookvale, Australia) in PD.ZSReleased nuclei were harvested and
quantitated in a Coulter counter (Coulter Electronics, Luton Beds,
England). The viability of cells present in the aspirated media and
in the washing buffer was determined by trypan blue dye exclusion.
Autoradiography. Highly purified, elutriated monocytes were
cultured in Lab-Tek eight-chamber tissue culture slides (1 cm2/
chamber; Miles Scientific, Naperville, IL) at a density of 10s
celldchamber in 0.3 mL complete aMEM for 90 minutes. Nonad-
1973
herent cells were removed by multiple washes. The adherent cell
monolayer was cultured in 0.3 mL complete aMEM containing 3%
HS with and without CSFs. After 5 days of incubation, cells were
pulsed with 'H-TdR (4 pCi/mL) for 24 hours and the cells fixed
with 10% buffered formol saline (BFS). The slide was subjected to
a quick immersion in Ilford K2 nuclear track emulsion (Ilford, Mt.
Waverley, Australia) at 42°C and exposed for 4 days at room
temperature. A standard developing procedure was employed2'
with the use of D19 developer (Kodak, Coburg, Australia) (3
min/lVC), Ilford rapid fixer (Ilford) (4 min/l6"C), and 10% BFS
(5 min/room temperature). The process was concluded by counterstaining the slide with hematoxylin. The labeling index was taken as
the percentage of cells with 60 or more grains per nucleus. A
minimum of 500 cells per culture well was evaluated under 4 0 0 ~
magnification. Results are expressed as the mean f SEM of
triplicate cultures per stimulus.
Preparation of platelet-poor plasma-denved HS. As described by
Musson? heparinized whole AB blood from normal donors
(Melbourne Red Cross Blood Bank) was centrifuged at 2,000 x g
for 30 minutes at 10°C to remove cell fractions and platelets. The
platelet-poor human plasma was collected and incubated with 20
mmol/L CaCI, at 37°C until coagulation occurred, thereby depleting any coagulation factors present. The clot was spun down
(2,000 X g, 30 minutes, 10°C) and removed. The platelet-poor
plasma-derived HS fraction was stored at -20°C until use.
Reagents. Highly purified recombinant human CSFs were obtained as gifts from the following: CSF-1, 4.2 x lo' U/mg
(Department of Process and Product Development, Cetus, Emeryville, CA)--CSF-l bioactivity was measured using C57B1/6
bone marrow cells in semisolid agar medium assigning 50 U/mL to
the concentration giving half-maximal colony formationz; bacterially synthesized GM-CSF, 5 x 10' U/mg (Dr J.F. DeLamarter,
Biogen, Geneva, Switzerland)-the bioactivity was assayed by day
14 colony growth of normal human bone marrow samples29;IL-3,2
to 4 x 10' U/mg (Dr G. Wong, Genetics Institute, Cambridge,
MA)-the bioactivity was measured in a proliferation assay using
primary cells from chronic myeloid leukemic patients assigning 1
U/mL to the concentration givinghalf-maximal stimulation. Recombinant human IFN-y (2.7 X 10' U/mg) was kindly provided by Dr
E. Hochuli, Hoffman-La Roche, Basel, Switzerland. IFN-y bioactivity was monitored using the human aminotic WISH cell viral
protection assay.MRecombinant human TNF-a (2.7 x lo7 U/mg)
was a gift from Dr G.R. Adolf, Ernst-Boehringer Institut, Vienna,
Austria. TNF-a bioactivity was assayed in the murine L-cell
cytotoxicity assay," with 1 U defined as the amount that caused
50% cytolysis. Recombinant human IL-4 (1 x 10' U/mg) was
obtained from DNAX, Palo Alto, CA. IL-4 bioactivity was measured in a proliferation assay using phytohemagglutinin-activated
T cells:' with 1 U/mL of activity defined to give half-maximal
cellular growth.
The following reagents were obtained commercially: 8-bromoadenosine 3':5'-cyclic monophosphate (8BrcAMP) and PGE, (Sigma),
cholera toxin (CT) (Calbiochem, La Jolla, CA), PT (List Biologicals, Campbell, CA), 'H-TdR (50 to 90 Ci/mmol) (Du Pont, NEN
Products, Boston, MA).
Extra precautions were taken to eliminate endotoxin contamination. Plastic disposable equipment was used whenever possible.
Glassware was soaked in 1% E-Toxa-clean (Sigma) and, after
multiple rinsings, baked at 240°C for 4 hours. All buffers and media
were filtered through Zetapor membranes (AMF Cuno, Meriden,
CT). All monocyte cultures contained polymyxin B sulfate at a final
concentration of 1 pg/mL. LPS levels of 10 pg/mL or less in all
reagents were confirmed in the limulus lysate assay (M.A. Bioproducts, Dynasciences, Walkermille, MD).
Statistical analysis of data. Where indicated, statistical signifi-
1974
CHEUNG AND HAMILTON
2
4
6
8
1
0
12
14
B
0
2
4
6
8
1 0 1 2 1 4
Days in culture
Fig 1. Effects of CSF-1 on DNA synthesis and cell number. Purified
monocytes from a typical donor were seeded in =-well plates and
allowed to adhere for 90 minutes. Nonadherent cells were removed
by multiple washes and the remaining adherent cell monolayers were
treated with 3% HS together with CSF-1 (3,000 U/mL) (0)or 3% HS
alone ( 0 )for up to 11 days. (A) Cells were pulsed with OH-TdR (2.5
pCi/mL) for the last 24 hours of incubation. At the time points
indicated, cultures were terminated and DNA synthesis was monitored by measuring the incorporation of %I-TdR as described in the
Methods. (6) In parallel cultures, the change in cell number was
determined at the time points indicated, which corresponded to the
end of the labeling period for measuring IH-TdR incorporation. For
both (A) and (6).each point represents the mean of triplicate cultures
(tSEM). Error bars have been omitted when errors were within the
size of the symbol.
100 pmol/L) increased the level of 'H-TdR incorporation
beginning between days 2 and 4, with the degree of
incorporation peaking between days 6 and 8. It is unlikely
that the decline at later time points was due to the
depletion of CSF-1, as the addition of fresh CSF-1 (3,000
U/mL) every second day did not maintain the level (data
not shown). When the increasing levels of 'H-TdR incorporation upon CSF-1 treatment were expressed on a per cell
basis, they were also significantly greater than that of the
control cultures, indicating that the increase of cellular
DNA synthetic activity was not due to the presence of more
cells in the CSF-l-containing cultures. The incorporation
of 'H-TdR into contaminating lymphocytes could not account for the increase detected, since the lymphocyteenriched fractions, obtained by elutriation, did not respond
to CSF-1 for DNA synthesis (data not shown). In agreement with previous finding in murine BMM," no DNA
synthesis was observed in serum-free cultures, even in the
presence of CSF-1 (data not shown).
In the same experiment, the effect of CSF-1 on cell
number was also examined. For control cultures incubated
with 3% HS alone, less than 50% of the initial count
remained adherent after 2 days of incubation (Fig 1B). For
the detached cells, approximately 30% of them were viable
and the percentage of viability decreased gradually with
time (data not shown). On the contrary, 80% of the initial
cell population in cultures treated with CSF-1 remained
adherent after 2 days. The number of adherent cells
increased between days 6 and 8; the original cell number
doubled by day 10. Similar kinetics were observed in two
other experiments. The addition of fresh CSF-1 every
second day did not enhance the increase (data not shown).
Increased 'H-TdR incorporation induced by CSF-1 was
dose-dependent (Fig 2). Monocytes attained maximal level
cance of measurements was determined by two-tailed, unpaired
Student's t test, where the level of significance was taken as P <
.05.
0
RESULTS
Stimulation of DNA synthesk and increase in cell number by
CSF-1. When cultured in 3% HS for 3 days, human
monocytes had a rounded appearance. In contrast, when
CSF-1 was added, the cells enlarged, spread, and formed
elongated cytoplasmic processes. To determine to what
extent monocytes respond to CSF-1 for DNA synthesis
when cultured under these conditions, 'H-TdR incorporation was measured over a 12-day period. The data in Fig 1A
are representative of four experiments. CSF-1(3,000 U/mL;
100
1000
10000
CSF-1 ( U / d )
Fig 2. Dose effects of CSF-1on DNA synthesis. Purified monocytes
from a representatlve donor were seeded in 24-well plates and
allowed to adhere for 90 minutes. Nonadherent cells were removed
by multiple washes and the remaining adherent cell populations were
incubated with CSF-1 at concentrations indicated (100 to 10.000
U/mL) for 4 days ( 0 )or 6 days ( 0 )and pulsed with 'H-TdR (2.5
pCi/mL) during the last 24 houn of incubation. Cells were harvested
and monitored for the levels of 'H-TdR incorporation as described in
the Methods. Each point represents the mean of triplicate cultures
(-cSEM). Error bars have been omitted when errors were within the
size of the symbol.
1975
HUMAN MONOCYTE DNA SYNTHESIS
of DNA synthesis in response to CSF-1 at doses greater
than or equal to 3,000 U/mL ( 2100 pmol/L).
GM-CSF, IL-3, and human monocyteproliferatwn. Other
CSFs, such as GM-CSF and IL-3, were assessed for their
ability to cause DNA synthesis in human monocytes cultured under the same conditions. The induction of DNA
synthesis by GM-CSF or IL-3 was dose-dependent (Fig 3).
GM-CSF exerted its maximal effect at doses of 100 U/mL
or greater (0.11 nmol/L) (Fig 3A), whereas for IL-3 it was
at 1 U/mL (0.05 nmol/L) as determined in a separate
donor (Fig 3B). It should be noted that 10 U/mL (0.5
nmol/L) of IL-3 was required in some donors to achieve
maximal effect. The relative effects of the CSFs on DNA
synthesis were compared. For cells from three of four
donors, CSF-1 enhanced 'H-TdR incorporation at a significantly greater degree than GM-CSF or IL-3 (P < .001) (Fig
4A). Any difference in the degree of response induced by
the growth factors was not detected in the other donor.
Control
,-1
u-3
CSF-1
0.4
A
-L
3
X
-----
3
Q
3
\
E
0.1
e
W
0.0
C
0
.r(
U
Control
G .
U-3
[Additions]
e
8
.s
GMCSF
7
4
0
.01
.1
1
10
100
Fig 4. Effects of CSF-1, OM-CSF, and IL-3 human monocyte DNA
synthesis and cell number. (A) Purified monocytes were seeded in
24-well plates and allowed t o adhere for 90 minutes. Nonadherent
cells were removed by multiple washes and the remaining adherent
cell monolayers were exposed t o 3% HS alone or together with either
CSF-1 (3,000 U/mL), GM-CSF (1.000 U/mL), or IL-3 (10 UlmL) for 5
days followed by a 24-hour labeling period. The levels of 'H-TdR
incorporation were determined as described in the Methods. ( 6 )
Monocytes purified from a different individual were prepared as
described in (A); the adherent cell populations were treated with
serum alone or together with either CSF-1 (3,000 U/mL), GM-CSF
(1.000 U/mL), or IL-3 (10 U/mL) for 11 days. The dashed line
represents the cell number at the time of addition of the various
growth factors (day 0). The change in cell number of the adherent
population was determined as described in the Methods. Relative t o
CSF-1-treated cultures: *P < .02, **P < .001. For both (A) and (6).
each point represents the mean of triplicate cultures (+SEM). Error
bars have been omitted when errors were within the size of the
symbol.
IL-3 (U/ml)
Fig 3. Effects of GM-CSF and IL-3 on DNA synthesis. Purified
monocytes from two separate donors were seeded in 24-well plates
and allowed t o adhere for 90 minutes. Nonadherent cells were
removed by multiple washes and the remaining adherent cell populations were treated with 3% HS alone or together with either (A)
GM-CSF (10 t o 1,000 UlmL) or ( 6 )IL-3 (0.01 t o 10 U/mL) for up t o 5
days and pulsed with IH-TdR (2.5 KCilmL) for 24 hours. At the end of
the labeling period, the levels of 3H-TdRincorporation were measured
as described in the Methods. .For both (A) and (E), each point
represents the mean of triplicate cultures (&$+EM).Error bars have
been omitted when errors were within the size of the symbol.
When changes in cell number were followed, all three
growth factors caused a significant increase in cell number
after 11days in culture (P < .Ol). However, in two of three
donors, CSF-1 treatment resulted in a greater degree of
increase, as shown in Fig 4B. Perhaps not surprisingly, the
proliferative response of human monocytes to any particular CSF is subject to donor-to-donor variation.
Number of 'H-TdR4abeled monocytes. In view of the
finding that CSF treatment was associated with increased
1976
CHEUNG AND HAMILTON
3H-TdR incorporation and cell number (Figs 1 to 3), the
proportion of cells that underwent DNA synthesis following CSF treatment was examined by autoradiography.
Five-day-old CSF-treated monocyte cultures were pulsed
with 'H-TdR (4 pCi/mL) for 24 hours. Table 1 depicts the
proportion of cells labeled with 60 or more nuclear grains in
four separate experiments. Approximately 9% to 16% of
monocytes were labeled in CSF-1-treated cultures within
the 24-hour labeling period, compared with the corresponding control value of 1% to 2.5%. In the GM-CSF-treated
and IL-3-treated groups, the percentage of labeled cells
varied from 5% to 10% and 7% to lo%, respectively. The
labeling index was not improved by lengthening the labeling
period (data not shown). It should be noted that the control
value may have been overestimated due to a decrease in the
absolute number of cells remaining in cultures (see above).
Synergistic effects of the CSFs. To determine whether the
CSFs could act synergistically, as has been reported for
murine BMM,% monocyte cultures were treated with a
suboptimal dose of either GM-CSF (5 U/mL; 5 pmol/L) or
IL-3 (0.1 U/mL; 5 pmol/L), together with different concentrations of CSF-1 (10 to 5,000 U/mL); 'H-TdR incorporation was measured at day 3. Both GM-CSF and IL-3
enhanced synergistically the levels of DNA synthesis by
monocytes treated with suboptimal doses of CSF-1 (100 to
1,000 U/mL) in three donors tested. Figure 5 shows the
response in one experiment; for cells from this particular
donor, the dose of GM-CSF administered induced a higher
level of 3H-TdR than IL-3 when these growth factors were
added alone to cultures. The more potent effect of GMCSF with cells from this particular donor may account for
the observation that GM-CSF potentiated the effect of
CSF-1 to a greater extent. It was also observed in this
particular donor that GM-CSF and IL-3 could potentiate
an optimal concentration of CSF-1 for the induction of
DNA synthesis. It is unlikely that the increase was due to an
earlier maximum level of DNA synthesis, since the presence of either GM-CSF or IL-3 did not alter the kinetics of
CSF-1-induced DNA synthesis (data not shown).
Effect of cytokines on CSF-l-induced DNA synthesis.
The in vitro development of human myeloid colonies from
bone marrow progenitor cells in the presence of CSFs can
be inhibited by IFN-y," IL-4," and TNF-a,I9while CSF-1stimulated murine BMM DNA synthesis can be suppressed
by IFN-y and TNF-(w." We therefore determined whether
Table 1. Number of 'H-TdR-Labeled Monocytes Treated with CSFs
Labeled Cells (% 2 SEM)
Treatment*
Control
CSF-1
GM-CSF
IL-3
Donor 1
* 0.07
0.9
16.3 k
9.5 k
9.2 2
1.5
2.2
1.8
Donor 2
Donor 3
Donor 4
2.5 2 0.5
10.9 f 0.4
5.2k 0.4
6.9 k 1
1.3* 0.1
9.5 f 0.3
6.6 k 0.5
6.7 2 0.2
1.7 1.3
9.2 f 1.5
9.6 f 0.9
10.2 2 1.3
*
*Purified monocytes were cultured in Lab-Tek eight-chamber slides,
and were treated with 3% HS or together with either CSF-1 (3,000
U/mL), GM-CSF (1,000 U/mL), or IL-3 ( I O U/mL) after a 90-minute
adherence period. The percentage of labeled nuclei was measured as
in the Methods. The results are expressed as the mean of triplicate
cultures SEM for four separate experiments.
Eli
m
0
IO
100
1000
10000
CSF-1 (U/ml)
Fig 5. Synergistic effects of the CSFs. Purified monocytes were
seeded in 24-well plates and allowed to adhere for 90 minutes.
Nonadherentcells were removed by multiple washes and the remaining adherent cell populations were stimulated with different concentrations of CSF-1 alone (10 to 5,000 U/mL) ( 0 )or together with either
GM-CSF (5 U/mL; 5 pmol/L) ( 0 )or IL-3 (0.1 U/mL; 5 pmol/L) (H)for
3 days, followed by a 24-hour labeling period. DNA synthesis was
measured as described in the Methods. Each point represents the
mean of triplicate cultures (SEM). Error bars have been omitted
when errors were within the size of the symbol.
these inhibitory cytokines could also reduce CSF-1stimulated human monocyte DNA synthesis.
In Fig 6A, CSF-1-treated cultures were exposed to
IFN-y (0.02 to 20 U/mL) for the entire 4-day culture
period. IFN-y at doses between 2 and 20 U/mL completely
abrogated the mitogenic effect of CSF-1, with the resulting
level of DNA synthesis lower than the basal. The inhibitory
effect of IFN-y could not be overcome by using higher doses
of CSF-1 (Fig 6B). This inhibitory effect of IFN-y was seen
in all three donors tested.
Cells from five donors were examined for the action of
IL-4 and representative results are presented in Fig 7.
Maximal inhibition of CSF-1-initiated DNA synthesis by
IL-4 was achieved at the doses of lo-" to lo-'' mol/L (Fig
7A); a supraoptimal dose of CSF-1 did not overcome the
suppressive effect of IL-4 (Fig 7B). This present finding
unveils another biological effect that IL-4 and IFN-y have
in common on human monocytes? although they can also
have opposite effect~.~~."
Unlike IFN-y and IL-4, whose
potent inhibitory effects on DNA synthesis were seen in all
donors, the suppressive effect of TNF-a on CSF-1-induced
DNA synthesis was partial (Fig 8A). TNF-a at the dose of
lo-* mol/L displayed a maximal inhibitory effect (Fig 8A);
its inhibitory action could be partially overcome by a
supraoptimal dose of CSF-1 in two of four donors tested, an
example of which is provided (Fig 8B).
It has been reported that IFN-y and TNF-a can be added
to CSF-1-stimulated murine BMM quite late in the GI
phase of the cell cycle and still achieve their maximal
inhibitory effects on DNA
Likewise, we demonstrate in Fig 9 that the administration of these cytokines,
as well as IL-4, can be delayed for up to 48 hours after
adding the CSF-1 to human monocytes and still can lower
the level of DNA synthesis to basal, 3H-TdR incorporation
again being measured by a 24-hour pulse at day 3. As
1977
HUMAN MONOCYTE DNA SYNTHESIS
CSF-1 (Table 2). When the doses of agents used exceeded
those indicated below, they were found to have a cytotoxic
effect over the overall 4-day culture period, as judged by cell
morphology and the number of viable cells (data not
shown).
A
0.8
-
0.6 -
DISCUSSION
0.4
-
0.2-
NO CSF-1
0
0
.01
.1
lo
1
100
Using highly purified human peripheral blood monocytes, we demonstrated that these cells responded to CSF-1
with a gradual increase in DNA synthesis and this enhanced
activity started to decline between days 6 and 8 in culture
(Fig 1). 3H-TdR incorporation increased up to %-fold upon
treatment with an optimal dose of CSF-1 (3,000 U/mL)
IFNY (U/ml)
J
-.f-
1.5
Noadditions
-
0.5
-12
0
-10
-11
-9
IL-4 10gFIl
0.0
0
10'
lo2
io3
lo4
lo5
CSF-1 ( U / d )
Fig 6. Effects of IFNy on CSF-l-induced DNA synthesis. (A)
Purified monocytes were seeded in 96-well plates. After an overnight
adherence period, cultures were subjected to gentle washes to
remove nonadherent cells. The remaining adherent cell populations
were treated with CSF-1 (3,000 U/mL) alone or together with
different doses of IFNy (0.02 to 20 U/ mL) for the entire culture period
(4 days). (B) Purified monocytes from a different donor were prepared
as described in (A). The adherent cell populations were incubated
with serial concentrations of CSF-1 alone (10 to 10,000 U/mL) ( 0 )or
together with IFNy (20 UlmL) (0).For both (A) and (B). 'H-TdR (0.5
pCi/0.2 mL) was added to cultures at day 3; cells were pulsed for 24
hours. The levels of 'H-TdR incorporationwere measuredas described
in the Methods. Each point represents the mean of triplicate cultures
(*SEM). Error bars have been omitted when errors were within the
size of the symbol.
0.6 0.5 0.4 0.7
0.3 0.2 0.1
-
0.0
I
0
10'
.
I
.-
.
lo2
lo3
lo4
lo5
CSF-1 ( U / d )
judged by the number of viable cells scored at the termination of cultures, there was no evidence that the antiproliferative actions of IFN-y, IL-4, or TNF-a could be explained
by cytotoxic effects.
CAMPand DNA synthesis. Agents that elevate intracellular CAMPhave been shown to inhibit murine macrophage
colony formation from progenitor cells,38as well as to block
the mitogenic action of CSFs on murine BMM." Table 2
shows the effect of CAMP-elevating agents, 8BrcAMP, CT,
and PGE,, on CSF-1-induced DNA synthesis in human
monocytes. All three agents (8BrcAMP [lo-' to
mol/
L], CT [lo-'* mol/L], and PGE, [lo-' to lo-' mol/L])
caused a partial suppression of the mitogenic action of
Fig 7. Effects of IL-4 on CSF-1-induced DNAsynthesis. (A) Purified
monocytes were seeded in 96-well plates. After an overnight adherence period, cultures were subjected to gentle washes to remove
nonadherent cells. The remaining adherent cell populations were
treated with CSF-1 (3,000 UlmL) alone or together with different
doses of IL-4 (1 x lo-" to 5 x lo-'' mol/L) for the entire culture
period (4 days). (B) Purified monocytes from a different donor were
prepared as described in (A). The adherent cell populations were
incubated with serial concentrations of CSF-1 alone (10 to 10.000
For both (A)
UlmL) (a)or together with 11-4 (5 x lo-'' mol/L) (0).
and (B), 'H-TdR (0.5 pCi/O.P mL) was added to cultures at day 3 and
cells were pulsed for 24 hours. The levels of 'H-TdR incorporation
were measured as described in the Methods. Each point represents
the mean of triplicate cultures (SEMI. Error bars have been omitted
when errors were within the size of the symbol.
1978
CHEUNG AND HAMILTON
though the degree of enhancement and the percentage of
cycling cells are lower, possibly reflecting the different
culture conditions.'~"
Our findings disagree with the recent conclusion of
Mufson" that CSF-1 does not enhance human monocyte
proliferation. Such discrepancy may be due to different
assay time points and duration of the labeling period.
Becker et aI@ reported that CSF-1 induced maturation
markers on human monocytes after 4 to 5 days in culture,
which coincided with the time at which the level of DNA
synthesis was maximal, as described above. It is unknown
whether the cycling cells that we have detected are included
in the population expressing these markers. However,
cycling cells could be less mature and they could be
differentiating in response to CSF-1; this phenomenon may
offer an explanation for the loss of responsiveness to CSF-1
with time, although other reasons can be given, for example, loss of CSF-1 receptors or production of negative
regulators for DNA synthesis.
It has been reported recently that GM-CSF and IL-3 not
only can maintain survival of human monocytes, but also
are weakly mitogenic for these cells.'" In our experiments,
7% to 10% of the population were capable of synthesizing
DNA when exposed to GM-CSF (1,000 U/mL) or IL-3 (10
U/mL) (Fig 3, Table l), as compared with 1% to 3%
reported in the other ~tudies.~'
When monocytes were
exposed to combinations of the CSFs, marked synergistic
effects for both GM-CSF and IL-3 for CSF-1-mediated
Fig 8. Effects of TNF-a on CSF-linduced DNA synthesis. (A)
Purified monocytes were seeded in S w e l l plates. After an overnight
adherence period, cultures were subjected t o gentle washes t o
remove nonadherent cells. The remaining adherent cell populations
were treated with CSF-1 (3,000 U/mL) alone or together with
different doses of TNF-a (lo-" t o lo-' mol/L) for the entire culture
period (4 days). (B)Purified monocytes from a different donor were
prepared as described in (A). The adherent cell populations were
incubated with serial concentrations of CSF-1 alone (10 t o 10.000
U l m L ) (0)or together with TNF-a (lo-' mol/L) (0).For both (A) and
(E), H-TdR (0.5 pCil0.2 mL) was added t o cultures at day 3 and cells
were pulsed for 24 hours. The levels of IH-TdR incorporation were
measured as described in the Methods. Each point represents the
mean of triplicate cultures (+SEM). Error bars have been omitted
when errors were within the size of the symbol.
(Figs 1 and 2). The addition of CSF-1 also resulted in an
increase in cell number (Fig 1B). The increase in cell
number in response to CSF-1 became apparent more than 2
days after maximal DNA synthesis. Therefore, there appears to be a prolonged period between the S phase and the
M phase of the cell cycle for the cycling subpopulation.
Autoradiography showed that approximately 10% of the
adherent population was found to undergo DNA synthesis
within a 24-hour labeling period at the time of maximal
DNA synthesis (Table 1). Others have also shown that
CSF-1 stimulates human monocyte DNA synthesis, al-
Fig 9. Effects of delayed addition of IFN-y, IL4, and TNF-a t o
CSF-1-treated cultures. Purified monocytes were seeded in %-well
plates and allowed t o adhere overnight. Nonadherent cells were
removed by gentle washes. Control cultures were incubated in 3% HS
alone. The rest of the cultures contained CSF-1 (3,000 U / mL) together
with serum. To some cultures, 1FN-y (100 UlmL), IL4 (5 x lo-'a
mol/L) or TNF-a (10.' mol/L) was added following the addition of
CSF-1 at the time points indicated (t). WTdR (0.5 pWO.2 mL) was
added t o all cultures at day 3 and cells were pulsed for 24 hours.
The levels of 'H-TdR incorporation were measured as described in the
Methods. The results are expressed as the mean of triplicate cultures 2 SEM. Error bars have been omitted when errors were within
the size of the symbol. Relative t o cultures treated with CSF-1 (3,000
U/mL) alone: *f < .001; +*P c .02; **+f< .05.
1979
HUMAN MONOCYTE DNA SYNTHESIS
Table 2. Effects of CAMP-Elevating Agents on CSF-l-Mediated DNA
Synthesis
~~
~
Treatment*
’H-TdR Incorporation
(cpmlwell -+ SEM)t
Control
CFS-1
8BrcAMP (10-6mol/L)
+8BrcAMP (lO-‘mol/L)
Control
CSF-I
CT (lo-’’ mol/L)
CT (lo-’’ mol/L)
Control
CSF-1
PGE, (lo-* mol/L)
PGE, (lo-’ mol/L)
1,000 2 70
8,600 f 420
7,200 2 70
4,800 f 140
2,000 f 170
30,000 2 1,800
32,900 f 330
16,400 2 700
430 f 20
10,500 2 54
4,200 f 500
2,200 f 370
+
+
+
+
+
PValueS
< .05
< .01
NS
< .01
< .02
< .02
The effects of 8BrcAMP, CT, and PGE, on CSF-l-mediated monocyte
DNA synthesis were examined separately in three different experiments. Data obtained from each experiment were compiled into this
Table.
‘Purified monocytes from three individuals were seeded in 96-well
plates and allowed to adhere overnight. Nonadherent cells were
removed by gentle washes. Control cultures were incubated in 3% HS
alone. The rest of the cultures contained CSF-1 (3,000 U/mL) together
with serum. To some cultures, a CAMP-elevating agent, 8BrcAMP. CT,
or PGE,, was added as indicated.
tCells were pulsed with ’H-TdR (2.5 pCi/mL) over the last 24 hours of
a 4-day culture. ’H-TdR incorporationwas meausred as described in the
Methods. The results are expressed as the mean of triplicate cultures 2
SEM.
SP values were determined by comparison with corresponding
cultures treated with CSF-1 alone. NS, not significant,P > .05.
DNA synthesis were observed (Fig 5). In some but not all
donors, the stimulatory effect of an optimal concentration
of CSF-1 could be further enhanced by GM-CSF or IL-3
(Fig 5). Whether there are different subpopulations of cells
which are selectively responsive to individual growth factors
remains to be elucidated. Synergistic interaction between
hematopoietic growth factors, when used at suboptimal
concentrations, have also been reported in murine cellsBMM? PEM, and monocytes.“ It is unknown whether
these synergistic interactions are a reflection of changes in
receptor properties, although it has been shown in murine
PEM that CSF-1 receptor expression is upregulated by
IL-3” and there is cross-talk between CSFs when acting on
murine progenitor cells.”
It was shown above that IFN-y, IL-4, and TNF-a inhibited the mitogenic action of CSF-1 on the monocytes (Figs 6
to 8). These findings parallel those with human marrow
progenitor cells which give rise to mature myeloid cells in
the presence of CSFS.”,’’,~.~~
They are also similar to the
action of IFN-y and TNF-a on murine BMM proliferation”
treated with CSFs, although there is controversy in this
system about the action of TNF-a.- It is known that
IFN-y and IL-4 induce cultured monocytes/macrophages
to undergo antigenic changes and formation of multinucleated giant cells (MGCs), which are indicative of cellular
m a t ~ r a t i o n . ~Whether
~ . ~ ’ the antimitogenic actions of IFN-y
and IL-4 as described above are related to the promotion of
early maturation is unknown. In our experiments, formation of MGCs in IFN-y- and IL-&treated CSF-l-containing cultures was evident (data not shown). Despite the
report that treatment of murine PEM with IFN-y resulted
in a decreased number of CSF-1 receptors,5’ and that
TNF-a caused a 90% downmodulation of CSF-1 receptors
in various murine macrophage populations,48it is unlikely to
be the explanation for the inhibitory actions of IFN-y, IL-4,
and TNF-a we have described. This is because of the
observations that CSF-1 was required only for the first 48
hours of a 4-day culture period to elicit a full-scale ’H-TdR
incorporation (data not shown), and yet the addition of the
antiproliferative cytokines at the end of this period was
shown to inhibit the activity of DNA synthesis to a substantial degree (Fig 9). It is more likely that the cytokines are
acting at the postreceptor level in the G, phase of the cell
cycle, possibly by the regulation of some critical event(s) in
the mitogenic signaling pathway(s) as has been proposed
for murine B M M . ~ ~
The present study demonstrated that 8BrcAMP, PGE,,
and CT all suppressed CSF-1-mediated DNA synthesis
(Table 2). Since PGE, and CT are able to increase
intracellular CAMPlevels in human monocytes,” as in many
cell types, these findings imply that CAMP-dependent
protein kinase A activation provides a negative signal for
human monocyte proliferation in response to CSF-1. These
observations are in keeping with others using human bone
marrow progenitor^,'^ murine BMM,” and a CSF-1dependent murine cell line.” Evidence was presented in the
latter two studies that the agents which elevated intracellular CAMP did not interfere with the interaction of CSF-1
with its receptor or with receptor function; whether this is
the case in human monocytes requires further investigation.
It is known that elevation of intracellular CAMP is associated with human monocyte differentiati~n.”,’~Thus, it
could be that the observed negative regulatory effects of the
CAMP-elevating agents and the cytokines, IFN-y, IL-4, and
TNF-a, are a result of cellular differentiation.
In support of an earlier observation made by Imamura et
a1,8 it was found that pretreatment of monocyte cultures
overnight with PT abolished the action of CSF-1 for the
induction of DNA synthesis (data not shown). The involvement of a PT-sensitive G-protein(s) in the induction of
signals from the occupied CSF-1 receptor to the initiation
of DNA synthesis in these cells is therefore likely.
The findings reported above indicate that a subpopulation of human monocytes can proliferate in response to
CSFs and various agents can be used to inhibit this growth.
It is possible that only a certain fraction of the monocytes
had time to undergo DNA synthesis during the assay
period, although, as indicated, lengthening the labeling
period did not increase the number of cells incorporating
’H-TdR. The actions of the various growth stimulators and
inhibitors are similar on human bone marrow progenitor
cells in vitro during the development of monocytes/
macrophages, although the difficulty in obtaining sufficient
numbers of enriched bone marrow progenitors makes
biochemical analysis awkward; on this basis, we would like
1980
to suggest that the active monocyte population, particularly
if it could be enriched further, might be a model cell type to
understand the molecular events controlling human myelopoiesis. The noncycling populations may represent a more
mature subpopulation. If these two putative subpopulations
could be separated, it might be possible to compare their
biochemical properties, and perhaps lead to a definition of
the differences between them. The net result might be a
better understanding of the events controlling human
myeloid cell proliferation and differentiation. The results
above also parallel those reported with murine macrophage
CHEUNG AND HAMILTON
populations," suggesting that the murine models may be
appropriatefor their human counterparts.
ACKNOWLEDGMENTS
We would like to thank Gin0 Vairo and Prue Hart for their
valuable discussions and Gino Vairo for reading the manuscript.
We also thank the following for generously providing cytokines:
Drs P. Ralph (recombinant human CSF-I), J.F. DeLamarter
(GM-CSF), G. Wong (recombinant human IL-3), G.R. Adolf
(recombinant human TNFa), E. Hochuli (recombinant human
IFN-y), and DNAX, Palo Alto, CA (recombinant human IL-4).
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