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Tumor Necrosis Factor-alpha Strongly Potentiates Interleukin-3 and
Granulocyte-Macrophage Colony-Stimulating Factor-Induced Proliferation of
Human CD34+ Hematopoietic Progenitor Cells
By Christophe Caux, Sem Saeland, Catherine Favre, Valerie Duvert, Patrice Mannoni, and Jacques Banchereau
Previous studies have shown that tumor necrosis factors
(TNFs) inhibit the proliferative effects of crude or purified
colony-stimulating factors (CSFs) on low density human
bone marrow cell fractions. In the present study we
investigated the effects of TNFa on the growth of highly
purified CD34' human hematopoietic progenitor cells (HPC)
in response to recombinant CSFs. In short-term liquid
cultures (5 t o 8 days), TNFa strongly potentiates interleukin3 (IL-3) and granulocyte-macrophage-CSF (GM-CSF)induced growth of CD34' HPC, while it has no proliferative
effect per se. Within 8 days, the number of viable cells
obtained in TNFa-supplemented cultures is threefold higher
than in cultures carried out with IL-3 or GM-CSF alone.
Secondary liquid cultures showed that the potentiating
effect of TNFa on IL-3-induced proliferation of CD34' HPC
does not result from an IL-3-dependent generation of
TNFa responsive cells. Limiting dilution analysis indicates
that TNFa increases both the frequency of IL-3 responding
cells and the average size of the IL-3-dependent clones.
The potentiating effect of TNFa on IL-3- and GM-CSFdependent growth of CD34' HPC is also observed in day 7
colony assays. Under these short-term culture conditions,
TNFa does not appear to accelerate cell maturation as a
precursor morphology is retained. Finally, TNFa inhibits
the relatively weak growth-promoting effect of granulocyteCSF (G-CSF), which acts on a more committed subpopulation of CD34' HPC different from that recruited by IL-3 and
GM-CSF. TNFB displays the same modulatory effects as
TNFa. Thus, TNFs appear t o enhance the early stages of
myelopoiesis.
0 1990 by The American Society of Hematology.
T
clear cells, contains most of the hematopoietic progenitors,
including multipotential self-renewing progenitors, as CD34'
HPC can fully reconstitute lethally irradiated baboons.25In
this context, we have investigated the effects of other growth
regulators on CD34' progenitors. In the present study, using
short-term proliferative assays of CD34' HPC, we found
unexpectedly that T N F a , which has no growth-promoting
effect per se, strongly enhances both IL-3- and GM-CSFdependent proliferation of CD34' HPC. We further demonstrated that the potentiating effect of T N F a on IL-3-induced
proliferation of CD34+ H P C is direct and the consequence of
both increased frequency and growth-rate of IL-3 responsive
cells.
H E IN VITRO proliferation of human hematopoietic
progenitor cells (HPC) depends upon the presence of
exogenous growth factors such as interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GMGSF). G-CSF, and M-CSF.' In contrast, soluble factors
such as interferons (I F N s ) , ~ transforming
.~
growth factor p
(TGF-@),5-7
and tumor necrosis factors ( T N F S ) ~ .have
' ~ been
shown to inhibit in vitro hematopoiesis, although their mode
of action is not well characterized.
T N F a is a protein produced by various cell types'' and
that was originally shown to induce necrosis of tumors in
vivo.I8 A cDNA clone coding for human T N F a has been
isolated,' and the availability of recombinant preparations
has revealed that this cytokine displays a broad spectrum of
activities (reviewed in reference 17). Among those activities,
T N F a , alone or in synergy with IFN-7, has been reported to
inhibit in vitro hematopoietic colony formation by granulocytic and monocytic,' 1-13.'6e r y t h r ~ i d , ' ~ - "and
. ' ~ multipotential progenitorsl0,I6when tested on total mononuclear bone
marrow cells or fractions thereof depleted of mature cells.1°-'6
Recently, we and other^'^-*^ have shown that IL-3, GMCSF, and G-CSF can induce the proliferation of purified
HPC expressing the CD34 antigen. This population, which
represents 1% to 2% of bone marrow low density mononuFrom the Laboratory for Immunological Research. ScheringPlough (UNICET) Dardilly; and INSERM U119 and Centre
Regional Anticancereux, Marseille, France.
Submitted January 8, 1990; accepted February 16, 1990.
C.C. is the recipient of a grant from the Fondation Mkrieux.
Lyon. France.
Address reprint requests to Christophe Caux. MD, ScheringPlough (UNICET), Laboratory for Immunological Research, 27
chemin des Peupliers, BP 11,69572 Dardilly, France.
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 1990 by The American Society of Hematology.
0006-4971/90/7512-0001%3.00/0
2292
MATERIALS AND METHODS
Hematopoietic factors. Recombinant human IL-326 (provided
by Dr S. Tindall, Scbering-Research, Bloomfield, N J ) and G-CSF
(provided by Dr N. Arai, DNAX, Palo Alto, CA) were purified to
homogeneity. Specific activities of IL-3 and G-CSF were 5 x 10'
U/mg and 2 x lo6 U/mg, respectively; one unit corresponding to
half maximal proliferative activity as determined by (methyl--'H)
thymidine (3H-TdR; specific activity 25 Ci/mmol; CEA Saclay,
France) uptake by human HPC in liquid cultures. IL-3 and G-CSF
were used at a saturating concentration of 10 ng/mL (50 U/mL)
and 25 ng/mL (50 U/mL), respectively. Human GM-CSFZ7(provided by Drs P. Trotta and T.L. Nagabhushan, Schering-Research)
was expressed in Escherichia coli and subsequently purified to
homogeneity. Specific activity was 2 x IO6 U/mg, as previously
defined.*' GM-CSF was used at a saturating concentration of 100
ng/mL (200 U/mL). Supernatant of Cos 7 cells transfected with the
cDNA of human TNFP was used at a concentration of 0.5%
(vol/vol), and mock supernatant derived from Cos 7 cells transfected
with unrelated cDNA was used as negative control (provided by Dr
N. Arai, DNAX, Palo Alto, CA). Recombinant purified T N F a was
purchased from Genzyme (Boston, MA) (specific activity 2 x lo7
U/mg). In most experiments, it was used at a saturating concentration of 25 ng/mL (500 U/mL).
Collection and fractionation of cord blood and bone marrow cells.
Umbilical cord blood and bone marrow samples were collected
according to institutional guidelines. Cord blood was collected
during normal full-term deliveries in glass vessels containing preservBlood, Vol 75, No 12 (June 15), 1990: pp 2292-2298
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2293
TNFa POTENTIATES PROLIFERATION OF CD34’ CELLS
results were expressed as mean counts per minute (cpm) standard
deviation (SD).
Progenitor assays in semisolid medium. Semisolid cultures in
methylcellulose were carried out by plating 5 x lo’ cells per tissue
culture grade 35 mm Petri dish (Corning, NY), in the presence of 1
IU purified recombinant human erythropoietin (Epo; Amgen, Thousand Oaks, CA; specific activity, 7 x lo4 IU/mg), as previously
described in detail.” Duplicate dishes were plated in each experiment, and after 7 days of incubation at 37OC and 5% CO,, colonies
(250 cells) were counted using phase-contrast microscopy.
Limiting dilution studies. Limiting dilution studies were done in
96-well round-bottom microtest tissue culture plates (Nunc) in
Iscove’s modified Dulbecco’s medium (IMDM) containing 30%
FBS,
mol/L 2-mercaptoethanol, 0.5% wt/vol BSA neutralized
with NaHCO, and L-glutamine and antibiotics as indicated above.
For each cell concentration used, ranging from 0.5 to 40.5 cells per
well, and for each factor tested, aliquots of 50 pL were seeded in 120
to 360 wells. After 5 to 7 days of culture (37OC, 5% CO,), the
positive wells were scored using an inverted microscope, and for
selected cell concentrations, the number of cells in each clone was
determined.
For calculation of clone frequencies, the data were plotted as the
log of the fraction of nonresponding wells versus the number of cells
plated per well. Linear regression analysis was applied to the data to
calculate the best slope.28Confidence intervals around the slope were
calculated using a form of the Student’s t test. Conformance of the
data to linearity was tested by using a x2 analysis: a x2 derivedprobability P > .05 indicating single-hit curves, thus demonstrating
activity on a single limiting cell type.
Average clone size was calculated as the ratio of (total cell number
enumerated in 120 wells seeded at 1.5 cells/well)/(number of
responding cells present in the 120 wells observed).28Calculation of
average clone size was done on triplicates of 120 wells.
ative-free grade-1 sodium heparin (Sigma, St Louis, MO) at a final
concentration of 20 IU/mL. Marrow was obtained by iliac crest
aspiration from adult bone marrow transplant donors free of
hematological disease. Light-density mononuclear cells were isolated by Ficoll-Hypaque gradient separation at a density of 1.077
g/mL. Mononuclear fractions were depleted of adherent cells by
overnight incubation at 37OC in RPMI 1640 medium (GIBCO,
Grand Island, NY) supplemented with 1% wt/vol tissue culture
grade bovine serum albumin (BSA; Boehringer Mannheim, FRG).
Cells bearing CD34 antigen were isolated from nonadherent mononuclear fractions through positive selection by indirect immune
“panning” using anti-My10 monoclonal antibody (MoAb) (HPCA1; Becton-Dickinson,Mountain View, CA), as reported
When the purity of the isolated cells, determined by FACScan
flow cytometric analysis, was below 95%, a second purification step
was performed using immunomagnetic beads. Briefly, after overnight incubation at 37OC, the CD34+ “panned” cells were resuspended at lo7 cells/mL in a cocktail of MoAbs (Leu-M3 [CD14],
Leu5b [CD2], Leu3 [CD4], Leulla [CD16], and Leu16 [CD20]
from Becton-Dickinson, at 2 pg/mL each) for 15 minutes, washed
twice, and incubated for 30 minutes with immunomagnetic beads
( lo7 beads/mL) coated with anti-mouse immunoglobulins (Dynabeads M450; Dynal, Oslo, Norway). The beads were removed using
a magnet, and the CD34’ cells were recovered in suspension. Thus,
in all experiments, the isolated cells were 95% to 99% CD34’, as
judged by staining with the anti-My10 MoAb.
Cell cultures in liquid medium. Cell cultures were established
in the presence of IL-3 (10 ng/mL) or IL-3 plus TNFa (25 ng/mL)
in medium consisting of RPMI 1640 supplemented with 10%
(vol/vol) heat-inactivated fetal bovine serum (FBS; Flow Laboratories, Irvine, UK), 10 mmol/L HEPES, 2 mmol/L L-glutamine, 5 x
10 -’mol/L 2-mercaptoethanol, penicillin (100 U/mL), and streptomycin (100 pg/mL); hereafter designated complete medium. CD34‘
cells were seeded in 24-well culture plates (Linbro; Flow Laboratories, McLean, VA) for expansion. Optimal conditions were maintained by splitting these cultures every 4 to 5 days. At different
timepoints as indicated, cells were collected for counting and
establishment of secondary cultures. For initiation of secondary
cultures, cells were washed 4 times and further incubated for 3 hours
in complete medium in order to eliminate growth factors. Subsequent proliferation assays were then carried out as described below.
For proliferative assays, cells were distributed in 96-well roundbottom microtest tissue culture plates (Nunc, Roskilde, Denmark)
at 5 x 10’ cells/100 pL complete medium and incubated (37°C. 5%
CO,) with saturating concentrations of factors for 6 days. After
incubation, cells were pulsed with 1 pCi of ’H-TdR for 6 hours,
harvested, and counted. Tests were carried out in triplicate, and
-
m
Fig 1. TNFa potentiates IL3-induced proliferation of highly
purified CD34’ HPC. (A) Kinetics of ’H-TdR uptake by CD34+
HPC cultured in IL-3 (10 ng/mL)
(-0-I# TNFa (25 ng/mL; -+-I,
IL-3
TNFa (-W-), or medium
alone (-0-1. (B) Viable cell
count kinetics of CD34’ HPC
cultured with IL-3 (-Ci-) or
IL-3
TNFa (-&I.
Cell numbers represent cumulative values from bulk cultures maintained in optimal conditions by
splitting on day 4 in the presence of factors.
RESULTS
TNFa potentiates IL-3-induced proliferation of CD34+
HPC. The effects of TNFa on CSF-induced proliferation
of C D 3 4 + HPC were examined in a liquid culture system.
Results shown in Fig 1A indicate that TNFa (25 ng/mL)
strongly enhances the IL-3-dependent proliferation of C D 3 4
HPC, whereas it has no proliferative effect per se. T h e
enhancement of ’H-TdR incorporation was observed at all
times tested. Figure 1B shows t h a t the enhanced ’H-TdR
incorporation (Fig 1A) reflects a n increased cell proliferation, since more viable cells a r e recovered in IL-3 plus TNFa
+
A
1001
+
25
+
1
0
2
’
1
4
.
1
6
days of culture
.
1
8
0
2
4
6
days of culture
8
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2294
CAUX ET AL
cultures when compared with IL-3 cultures. After 4 days and
8 days, approximately two- and threefold as many viable
cells, respectively, were recovered when T N F a was added to
cultures. The TNFa-induced enhancement of IL-3-mediated cell proliferation is dose dependent (Fig 2), with
maximal stimulation observed at approximately 2.5 ng/mL
TNFa. Interestingly, a combination of 2.5 to 25 ng/mL
T N F a with a half maximal dose of IL-3 (0.25 ng/mL)
results in a proliferative response higher than that obtained
with an optimal dose of IL-3 used alone (2.5 ng/mL).
TNFa and @ enhance the proliferation of CD34+ HPC
induced by IL-3 and GM-CSF, but inhibit that induced by
C-CSF. As GM-CSF and, to a lesser extent, G-CSF are
also able to induce the proliferation of CD34+ HPC,2"-22.24
we
wondered whether T N F a would also enhance cell proliferation induced by these factors. Thus, CD34+ HPC were
cultured with optimal concentrations of various factors used
alone or in dual combinations. Data shown in Table 1
indicate that T N F a is able to enhance the GM-CSFdependent proliferation of CD34' HPC, and, as observed
with IL-3, T N F a approximately doubles the level of 3H-TdR
incorporation. The recovery of viable cells is also enhanced
(data not shown). In contrast, T N F a inhibits the G-CSFdependent proliferation of CD34+ HPC. Finally, TNFB
displays the same activities as T N F a on CSF-induced
proliferation of CD34+ HPC.
IL-3 does not induce the generation of cells proliferating
in response to TNFa. Since we previously showed that
preculturing CD34+ HPC with IL-3 results in a subsequent
enhancement of the proliferative responses to GM-CSF and
G-CSF,"." we wondered whether IL-3 would induce the
appearance of a cell population proliferating in response to
T N F a alone. Thus, CD34+ HPC were precultured for
various periods of time with IL-3, and proliferative responsiveness to T N F a and CSFs was subsequently assayed as
measured by 'H-TdR incorporation in secondary liquid
h
r?
0
7
X
0
Y
m
c
P
=I
10-2
10-1
100
101
102
TNF ngiml
Fig 2 . Dose dependence of the potentiating effect of TNFa on
IL-3-induced CD34+ HPC proliferation. CD34' HPC ( 5 x lo3)were
cultured in TNFa (-+-I, TNFa 25 ng/mL IL-3 I-.-).
TNFa
2.5
ng/mL IL-3 (-0-1, TNFa
0.25 ng/mL IL-3 (-A-)*or TNFa
0.025 ng/mL IL-3 (-A-1. 3H-TdR uptake was measured on day 6 Of
culture.
+
+
+
+
Table 1. Effects of TNFs in Combination With CSFs on the
Proliferation of CD34' HPC in Liquid Culture
~
Factors
Medium
TNFa
Medium
IL-3
GM-CSF
G-CSF
3.5 t 1.0
53.6 t 6.8
20.2 t 2.5
10.5
1.9
5.5
2.8
95.2 i 11.4
41.9 t 4.2
5.1 t 2.1
*
*
TNFO
4.5 i 0.6
91.7 k 7.2
40.9 6 . 8
0.4
4.9
*
*
~
IL-3
53.6
53.6
66.1
63.0
t 6.8
k
6.8
t 3.8
t 3.8
Values show 3H-TdR uptake (CPM x
k SD) obtained from one
representative experiment of three and expressed as mean t SD of
triplicate wells. Proliferation was measured at day 6 ; TNFs and all CSFs
were used at saturating concentrations, as described in Materials and
Methods.
cultures. Results shown in Fig 3 indicate that T N F a alone
has no direct proliferative effect on IL-3 precultured cells,
while the cells remain responsive to IL-3, GM-CSF, and
G-CSF. The same results were obtained when preculturing
CD34+ HPC with IL-3 plus T N F a (data not shown).
TNFa increases the frequency and size of IL-3 responsive
clones. We next addressed whether the potentiating effect
of T N F a on IL-3-dependent cell growth was due to an
increased frequency of IL-3 responding cells, to an enhanced
growth rate of cells already responding to IL-3, or to both. To
answer this question, clonogenic day 7 CFU assays in
methylcellulose and limiting dilution studies in liquid medium were carried out with CD34+ HPC.
First, day 7 CFU, from cord blood or bone marrow CD34+
HPC (Table 2) show that T N F a enhances the total number
of colonies induced by IL-3 and GM-CSF, but blocks the
G-CSF-dependent generation of colonies. In addition, colonies generated in response to combinations of IL-3 and
T N F a or GM-CSF and T N F a were larger than those
generated in the presence of IL-3 or GM-CSF alone (not
shown).
Second, limiting dilution studies were done where CD34+
HPC were seeded at various concentrations into wells of
microtitration plates with IL-3 and with or without TNFa.
The number of growing clones was determined after 6 days of
culture. As shown in Fig 4, T N F a increases the frequency of
IL-3 responding cells (1 of 3.2 cells generates clones in the
presence of IL-3 and T N F a , whereas 1 of 4.5 cells generates
clones in the presence of IL-3 alone). The combination of
1L-3 and T N F a generates 20% to 40% more clones than IL-3
alone (three separate experiments). The fact that the potentiating effect of T N F a is seen at very low cell concentration
(0.5 cells per well) and the linear slope of the frequency
analysis curves demonstrates that T N F a acts directly on the
CD34 ' HPC. We further determined the number of cells in
positive wells (seeded with 1.5 cells) to estimate clone size
(Fig 5). Cultures done in the presence of T N F a and 1L-3
yielded a higher number of large clones (greater than 32
cells) and a lower number of small clones (less than 32 cells)
than those done with IL-3 alone. This is illustrated by a shift
of clone size distribution towards the larger size clones (Fig
5) and by a higher average clone size in the combination of
IL-3 plus T N F a (59 cells per clone, experiment 1 and 53 cells
per clone, experiment 2) than in IL-3 alone (34 cells per
clone, experiment 1 and 32 cells per clone, experiment 2).
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2295
TNFa POTENTIATES PROLIFERATION OF CD34' CELLS
T
40
30
Fig 3. The potentiating affect of TNFa on
IL-3-induced proliferation of CD34+ HPC is not a
consequence of an IL-3-dependent induction of
TNFa responding cells. Purified CD34' HPC were
precultured with IL-3 (10 ng/mL); and at various
time points, cells were harvested, washed, processed as described in Materials and Methods, and
subsequently assayed for secondary proliferative
GM-CSF ( 0 ) .G-CSF
responsiveness t o IL-3
(E@, TNFa (a),or medium alone (0).Responsiveness was measured by 3H-TdR uptake, 6 days after
initiation of secondary cultures.
20
10
(m),
0
0
12
5
days of pre-culture in IL-3
DISCUSSION
This study demonstrates that tumor necrosis factors
strongly potentiate the proliferative effects of IL-3 and
GM-CSF on CD34' HPC isolated from cord blood and bone
marrow. Within 8 days, the number of cells generated in
TNFa-supplemented liquid cultures of CD34+ HPC is three
times higher than in cultures carried out with IL-3 alone.
This reflects a true potentiating effect as T N F a per se does
not act as a growth factor for CD34+ HPC. In our hands,
T N F a appears to be the most potent activator of IL-3dependent proliferation of CD34+ HPC, since combinations
of IL-3 with GM-CSF, G-CSF, IL-1 (data not shown), and
IL-6 (data not shown) are always less potent than the
combination of IL-3 and TNFcu. As expected, TNFP, which
uses the same receptor as TNFa," also potentiates IL-3induced proliferation of CD34' HPC.
As shown by secondary liquid cultures, the observed
potentiation is not the consequence of an IL-34ependent
generation of cells proliferating in response to T N F a alone.
Rather, colony assays and limiting dilution studies indicate
that the effect of T N F a results from both an increased
frequency and an increased growth rate of IL-3 responding
cells. Furthermore, limiting dilution analysis demonstrates a
direct effect of T N F a on its target cells. Although T N F a
strongly potentiates the proliferative effects of IL-3, no
morphologic differences were observed in terms of cell
maturation between day 7 colonies generated with IL-3 and
those generated with IL-3 and T N F a (data not shown). In
number of cells / well
0
5
10
15
20
VI
E
0
e
.-
E
F
.-
0
Table 2. Effects of TNFa in Combination With CSFs on the
Induction of Day 7 CFU From Cord Blood and Bone Marrow
CD34+ HPC
0
n
E
c
0
Factors
Experiment 1
Medium
TNFa
Experiment 2
Medium
TNFa
Medium
IL-3
GM-CSF
G-CSF
L
c
0
I-
0
0
0
0
24 1
245
335
343
169
141
259
251
20
24
0
0
4
8
7
2
240
222
310
154
134
258
276
21
15
2
1
356
Duplicate dishes plated with 5 x lo3 cells in the presence of Epo (1
U/mL) and CSFs with or without TNFa (25 ng/mL) or in medium alone
were scored for number of colonies on day 7 (colonies 5 5 0 cells). For
CSF concentrations, see Materials and Methods. Experiment 1, cord
blood; Experiment 2, bone marrow.
Y
2
Fig 4. TNFa increases the frequency of IL-3 responsive CD34'
HPC. Replicate wells (1201 were seeded at 13.5 cells per well,
(2401 a t 4.5 cells per well, and (3601a t 1.5 and 0.5 cells per well.
The presence of clones was assessed on day 6. Single-hit kinetics
were obtained, and the slopes were drawn by linear regression
analysis. The shaded region represents the 95% confidence limit.
The clone frequencies obtained in this experiment were 1:4.5 =
0.22 ( P = .65) for IL-3 ( 4 - 1 and 1:3.2 = 0.31 ( P = .45) for IL-3
TNFa (-0-1. TNFa or medium alone did not induce the development of any clones. The experiment presented here is representative of three.
+
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2296
5
u)
CAUX ET AL
151
T
10
.c
0
z
P
E
5
3
c
0
8-15
A
16-31
32-63
64-127128-255
number of cells per clone
I
T
*
0
t
P
E
3
c
8-15
B
16-31
32-63
64-127 128-255
number of cells per clone
Fig 5. TNFa increases the size of IL-3-dependent clones.
Clone size distribution obtained in limiting dilution experiments
done with CD34' HPC seeded at 1.5 cells per well in the presence
of IL-3 (.I or 11-3 TNFa (@I. After 6 days of culture, the size of
each clone was determined by counting using an inverted microscope. TNFa or medium alone did not induce the development of
any clones (28 cells per well). The clones were classified into
sections of arbitrarily defined numbers. The numbers plotted
correspond to the mean of triplicates of 120 wells observed. The
average clone size calculated on triplicates of 120 wells were (A):
Experiment 1 , 3 4 k 3 for IL-3 and 59 k 4 for IL-3
TNFa: and ( 8 )
Experiment 2.32 k 4 for IL-3 and 53 + 2 for IL-3
TNFa.
+
+
+
this context, cells generated in short-term liquid culture (8
days) in the presence of T N F a and IL-3 express a phenotype
and a precursor morphology similar to that of cells generated
with IL-3 alone (data not shown). T N F a was also shown to
potentiate GM-CSF-induced proliferation of CD34+ HPC
in liquid or semisolid cultures with parameters comparable
with those observed with IL-3. In contrast, T N F a displays an
antagonist effect on the proliferation of CD34+ HPC induced
by G-CSF. In this context, we and other investigator^^^^^'.^^.^^
have previously shown that GM-CSF and IL-3 recruit
largely overlapping CD34 + cell populations, including multipotential progenitors, while G-CSF acts on a subpopulation
of CD34' committed progenitors. Thus, the potentiating and
antagonistic effects of T N F a on CSF-induced CD34+ HPC
proliferation may be related to the heterogeneity of C S F
responding target cells, which remain to be phenotypically
defined.
T N F a has generally been described as an inhibitor of in
vitro hematopoiesis.8,'' However, such studies reported that
the degree of inhibition mediated by T N F a was variable14.''
and depended on the cellular composition of the bone marrow
fractions tested and on the type of conditioned medium used
as the source of CSFs. These inhibitory effects of T N F a may
be indirect through the release of factors by accessory cells,"
factors that may act directly or in concert with T N F a to
inhibit hematopoie~is.~-'~*''
Thus, the use of heterogeneous
bone marrow cell fractions or conditioned medium as a
source of CSFs3' in previous studies could explain the
apparent discrepancies with our current results, obtained
with recombinant factors and CD34' HPC, a population
that is highly enriched for progenitor cells and devoid of
accessory cells. In keeping with the generally described
inhibitory effects of T N F a , we have found (in other experiments to be submitted) that T N F a can block cell growth in
long-term cultures (14 to 35 days) of CD34' HPC, when
well-differentiated cells are present in the cultures.
Our data on the potentiating effect of T N F a in short-term
cultures are supported by the recent observations that T N F a
can enhance GM-CSF-induced in vitro growth of cells from
some acute myeloid leukemia (AML)
As
previously shown for IL-3 and GM-CSF,20,33
such observations further indicate similarities in cytokine responsiveness
of AML cells and normal hematopoietic cells.
Additional studies are required to determine the mechanism of the potentiation observed in our present investigation. As T N F a has been shown to upregulate the expression
of the IL-2 receptor on activated T cells,34it is tempting to
speculate that T N F a might upregulate the expression of
IL-3 and GM-CSF receptors on CD34+ HPC. However,
T N F a may also mediate its effect by activating a specific
second messenger, thus amplifying mitotic signals given by
IL-3 or GM-CSF alone. Conversely, the inhibitory effect of
T N F a on G-CSF-dependent proliferation of CD34+ HPC
may result from downregulation of the G-CSF receptor or
from a blocking of intracellular pathways activated by
G-CSF.
TNFa, primarily identified as a cytotoxic factor was later
demonstrated to display growth-stimulatory properties on
different cell type^.^^-^' Such a capacity to display both
proliferative and antiproliferative effects is indeed shared by
many factor^.'^ In this study, we showed that TNFa,
originally described as an inhibitor of hematopoiesis, strongly
enhances the early stages of myelopoiesis. This finding
should be taken in consideration when clinical use of cytokines is proposed in order to control the proliferation of
leukemias or tumors.
ACKNOWLEDGMENT
We are grateful to N. Courbiere, M. Vatan, and C. MCnCtrier for
editorial assistance; 1. Durand for flow cytometric analysis; Dr J.P.
Favier, Professor R.C. Rudigoz, and colleagues for providing umbilical cord blood samples; Professor P. HervC and colleagues for
providing bone marrow samples; and Dr J.A. Waitz for support and
for critical reading of the manuscript.
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2297
TNFa POTENTIATES PROLIFERATION OF CD34+ CELLS
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1990 75: 2292-2298
Tumor necrosis factor-alpha strongly potentiates interleukin-3 and
granulocyte-macrophage colony-stimulating factor-induced
proliferation of human CD34+ hematopoietic progenitor cells
C Caux, S Saeland, C Favre, V Duvert, P Mannoni and J Banchereau
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