1. Art and Diseño

From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
Tumor Necrosis Factor (TNF)-a Directly Inhibits Human Erythropoiesis In
Vitro: Role of p55 and p75 TNF Receptors
By Leiv S. Rusten and Sten Eirik W. Jacobsen
is mainly mediated through TNFR-p55, although TNFR-p75Two tumor necrosis factor receptors (TNFRs)
with molecular
mediated inhibition could be observed on progenitors reweights of55kD(TNFR-p55)and75kD(TNFR-p75)have
sponsive to erythropoietin alone. Moreover, at low TNF-a
recentlybeenidentifiedandcloned.Inpreviousstudies,
concentrations (2 ng/mL), TNF-a stimulates interleukin-3TNFR-p55 has been shown to exclusively mediate bidirectional effects of TNF-a on committed bone marrow granulo- dependent in vitro growth of committed granulocyte-macrophage progenitor cells, whereas it potently inhibits erycyte-macrophage progenitorcells, whereas both TNFR-p55
throid progenitor cell proliferation, showing that one conand TNFR-p75 can mediate inhibition of primitive progenicentration of TNF-a can simultaneously and bidirectionally
tors requiring multiple cytokines to proliferate. We show
modulateinterleukin-3-dependentgrowth
of committed
here that TNF-a potently and directly inhibits the in vitro
granulocyte-macrophage(stimulation)anderythroidprogrowth of committed erythroid progenitor cells
in response
genitor cells (inhibition).
to multiple cytokine combinations, and
that TNF-a-induced
0 7 9 9 5 by The American Societyof Hematology.
inhibition of burst-forming unit-erythroid colony formation
Furthermore, because TNF-a has been implicated in the
UMOR NECROSIS FACTOR-a (TNF-a),a multifuncpathogenesis of the anemia associated with chronic distional cytokine shown to affect a number of different
it is important to determine which of the two TNFRs
cell types,I4 is mainly produced by activated macrophages
mediates this effect.
and lymphocyte^.^.^ In hematopoiesis, TNF-a acts as both a
Whereas TNF-a has been shown to have potent antitumor
positive and negative regulator of myeloid cell proliferation
activity,' its clinical use has been hampered by severe toxic
and differentiation."" The effects of TNF-a can be mediated
side effect^.“^ The recent development of TNF-a mutants
either directly'2,'6 or indirectly by inducing other cells to
with selective activity on either TNFR-p55 or TNFR-p75 has
produce hematopoietic growth factor^.'^.'^ Furthermore,
opened the potentiality of reducing the serious side effects of
TNF-a can modulate the expression of c-ki?' as well as cell
TNF-a while maintaining the anticancer effect.M4a
surface receptors for the colony-stimulating factors (CSFs)
The present study was designed to determine the direct
on both normal bone marrow (BM) cells and acute myeloid
effects of TNF-a on erythroid colony formation in response
leukemia cells.'632'-26
to growth factor combinations not previously explored for
Several previous studies have demonstrated inhibitory efwith TNF-a. This is of particular importance
fects of TNF-a on erythroid colony f o r m a t i ~ n . ~ ~ HOW" ~ ' ~ ~ ~ interactions
'~~~
because the effects of TNF-a (stimulatory or inhibitory) on
ever, many of these studies were performed in systems not
granulocyte-macrophage progenitors have been demonallowing segregation of direct and indirect effects of TNFstrated to be strictly dependent on the specific growth factor
a. Even more importantly, the interactions between TNF-a
stimulating proliferation."-13." Using agonistic anti-TNFR
and specific hematopoietic growth factors in erythropoiesis
antibodies, as well as TNF-a mutant proteins specific for
have only been examined to a limited extent.
either TNFR-p55 or T N F R - P ~we
~ ,also
~ ~ examined the relaTwo TNF receptors (TNFRs) with molecular weights of
tive role of the two TNF receptors in mediating TNF-a
55 kD (TNFR-p55) and 75 kD (TNFR-p75) have recently
effects on erythropoiesis.
been identified and ~ l o n e d . TNFR-p55,
~ ~ . ~ ~ expressed on a
majority of cell types, has been shown to mediate most efMATERIALS AND METHODS
fects of TNF-a, such as cytotoxicity, proliferation of fibroHematopoietic growth factors and antibodies. Purified recombiblasts, and prostaglandin ~ y n t h e s i s . Whereas
~ ~ . ~ ~ effects of
nant human (rHu) stem cell factor (SCF) was generously supplied
TNF-a mediated through TNFR-p75 appear to be much
by Dr Ian K. McNiece (Amgen Inc, Thousand Oaks, CA). rHuILmore restricted, it has been shown that the TNFR-p75 can
3 was generously provided by Dr Steven Gillis (Immunex Corp,
signal stimulation of T-cell p r o l i f e r a t i ~ n . Also,
~ ~ . ~ ~TNFRSeattle, WA). rHuIL-9 was a gift from Genetics Institute (Camp75 has been suggested to be involved in ~ y t o t o x i c i t y . ~ ~bridge,
~ ~ ~ MA), rHu-erythropoietin (Epo) was purchased from Cilag
We have recently found a differential role of the two TNFRs
AG (Schaffhausen, Switzerland). rHuTNF-a, TNF-a mutant proin murine hematopoiesis in that the p55 TNFR exclusively
mediates the effects of TNF-a on committed granulocytemacrophage progenitor cells, whereas the p75 TNFR is esFrom the Department of Immunology, Institute for Cancer Resential in signaling inhibition of primitive progenitors."' Simsearch, The Norwegian Radium Hospital, Oslo, Norway.
ilarly, on human BM progenitor cells, we have shown that
Submitted May 20, 1994; accepted October 18, 1994.
Supported by The Norwegian Cancer Society.
the potentiating effect of TNF-a on granulocyte-macrophage
Address reprint requests to Leiv S. Rusten, MD, Department of
colony-stimulating factor (GM-CSF)- or interleukin-3 (ILImmunology, Institute for Cancer Research, The Norwegian Radium
3)-induced proliferation as well as the inhibition of granuloHospital Hospital, N-0310 Oslo, Norway.
cyte colony-stimulating factor (G-CSF)-stimulated colony
The publication costs of this article were defrayed in part by page
formation is mediated exclusively through TNFR-p55."
charge payment. This article must therefore be hereby marked
However, both TNFR-p55 and TNFR-p75 are involved in
"advertisement" in accordance with 18 U.S.C. section I734 solely to
mediating growth inhibition of primitive high proliferative
indicate this fact.
potential colony-forming cells." However, the relative role
0 1995 by The American Society of Hematology.
of the two TNFRs in erythropoiesis has not beenestablished.
0006-4971/95/8504-0025$3.00/0
T
Blood, Vol 85, No 4 (February 15), 1995: pp 989-996
989
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
RUSTEN AND JACOBSEN
990
teins. and anti-TNFR antibodies were a kind gift from Drs W. Lesslauer and H. Loetscher (Hoffmann-LaRoche, Basel, Switzerland).
HuTNF-a mutants specific for TNFR-p55 and TNFR-p75 have been
prepared by site-directed mutagenesis.." Solid-phase binding studies
have shown that the Trp"ThrS6 TNF-a mutant protein binds with
wild-type affinity to TNFR-p55 and does not bind at all to TNFRp75, whereas the Asn"'Arg'JS TNF-a mutant protein exclusively
bindsto TNFR-p75, although with a S- toIO-fold lower affinity
than wild-type TNF-cY.~'
Both mutant proteins were titrated and used
at optimal concentrations as previously described." Rabbit antihuman TNFR-p75 polyclonal antibodies with TNF-a agonistic activity
(paTNFR-p75) and a monoclonal antibody (MoAb) against the
TNFR-p55 with TNF-a agonistic activity (htr-9) were raised and
prepared as previously described.Jg.5"Htr-9 and paTNFR-p75 were
initially titrated as described elsewhereI5and thereafter used at optimal concentrations of I O pglrnL (htr-9) and 2yglmL (paTNFRp75). Unless otherwise indicated, all growth factors wereusedat
predetermined optimal concentrations: rHulL-3 (20 ng/mL), rHuSCF
(SO ng/mL). rHulL-9 ( 1 0 0 IUlmL). and Epo ( 5 IUlmL).
Cell separation. Human BM cells were obtained by iliac crest
aspiration from normal adult volunteers with informed consent and
the approval of the Ethics Committee of The Norwegian Radium
Hospital. Mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation (Lymphoprep; Nycomed, Oslo, Norway). Positive
selection of CD34' cells was performed according to a previously
described method."'Briefly,BM
mononuclear cells were rosetted
with Dynabeads M-450 directly coated with the CD34 MoAb BI3C5 (Product No. 11 1 . 1 0 Dynal, Oslo, Norway) for 45 minutes at
4°C on an apparatus that provided tilting and gentle rotation. The
bead to total cell ratio was 1: I . Rosetted cells were attracted to a
samarium cobalt magnet and nonrosetting cells were removed by
pipetting and washed seven times. Detachment of beads from positively selected cells was performed by incubation with anti-Fab antiserum (DETACHaBEAD; Dynal) at a concentration of 35 mglmL
for I hour at room temperature. Isolated cells, free of beads, were
washed and counted. The purityofCD34'
cells isolated by this
method was reproducibly greater than 90% as determined by flow
cytometric analysis.
To enrich and separate committed erythroid (burst-forming uniterythroid [BFU-E]) and granulocyte-macrophage (colony-forming
unit-granulocyte-macrophage [CFU-GM]) progenitors, we took advantage of the differential expression of CD45 isoforms previously
described onthese cells?' Because all BFU-E are CD45RO' and
the majority of CFU-GM are CD45RO-, CD34TD45RO' cells are
enriched in committed erythroid progenitors, whereas committed
granulocyte-macrophage, but not erythroid, progenitors reside in the
CD34TD45RO- subpopulation. In addition, we wanted to deplete
our test cell populations in primitive progenitors. which was obtained
by selecting progenitors expressing CD38."." To isolate cells with
the CD34*CD38'CD45RO- or CD34'CD38'CD45RO- phenotypes by cell sorting, positively selected CD34' cells with a mean
purity (n = 3) of 94% as assessed by flow cytometry were stained
with anti-CD45RO (UCHL-I; Becton Dickinson, San Jose, CA)
directly conjugated to phycoerythrin (PE) andwith
anti-CD38
(IOB6; Immunotech, Marseilles, France) directly conjugated to fluorescein isothiocyanate (FITC). Isotype-matched RTC- and PE-conjugated irrelevant mouse MoAbs served as controls. Cell sorting was
performed on an Epics Elite cell sorter (Coulter Electronics, Hialeah,
FL). A sort gate within a dual-parameter cytogram of forward light
scatter against 90" side scatter was drawn. A second amorphous gate
was drawn on two-color cytograms, and sort equations were set to
positively sort cells satisfying both gates.
Colonv assay. CD34' cells were plated in a volume of 1 mL
Iscove's modified Dulbecco's medium (IMDM; GIBCO, Paisley,
UK) containing 20% fetal calf serum (FCS; Sera-lab, Sussex, UK),
o w
120
m
z
L
m
IL-3+Epo
IL-3+SCF+Epo
100
R
80
SCF+Epo
IL-P+Epo
60
40
20
n
" - + - +
- + - +
- +
TNF-a
Fig 1. Effects of TNF-a on BFU-E colony formation ofCD34' human BM cells. CD34' cells were isolated and plated in methylcellulose
as described in the Materials and Methods at 2 x lo3 cellslplate,
with predetermined optimal concentrations of the growth factors
indicated, in the absence or presence of TNF-a (100 nglmL with IL3 + SCF + Epo and 200 nglmL with SCF + Epo; otherwise, 2 ngl
mL). Cultures were scored for BFU-E colony growth after 14 days of
incubation at 37°C and 5% CO2 in air. Results are presented as t h e
mean number of colonies per 2 x lo3cells from at least four independent experiments with triplicate determinations; error bars show t h e
SEM. *No colony formation.
I .2% methylcellulose (Methocel 4,000 mPa-s; FlukaAG. Buchs.
Switzerland), 5 X IO-' mol/L 2-mercaptwthanol, 300 mg/L glutamine, 66 mg/L penicillin, 100 mg/L streptomycin, and recombinant
human growth factors as indicated. After 2 weeks of incubation at
37°C and 5% CO2 in air, colonies (>40 cells) derived from CFUGM and BFU-E were assessed according to established criteria'"
and counted using an inverted microscope. SCF-induced clusters (4
to 40 cells) were scored after I O days of incubation.
Single-cell proliferation assay. Test cells were seeded in Terasaki plates (Nunc, Kamstrup, Denmark) at a concentration of I cell
per well (300 wells per group) in 20 pL IMDM containing 20%
FCS (Sera-lab), L-glutamine, penicillin, and streptomycin. Wells
were scored for erythroid colony formation (>40 cells) after 2 weeks
of incubation at 37°C and 5% CO' in air. To verify the erythroid
composition of colonies induced by SCF + Epo. morphologic examination of randomly picked colonies was performed on May-Griinwald-Giemsa-stained cytocentrifuge cell preparations.
Statistical analysis. All results were expressed as themean 2
SEM of data obtained from three or more separate experiments.
The statistical significance of differences between group means was
determined using the Student's t-test.
RESULTS
Effects of TNF-a on erythroid colonyformation by human
BM CD34' cells. Weand others have previously shown
that TNF-a potently and directly can enhance granulocytemacrophage colony formation of human CD34' progenitors
stimulated by IL-3 or GM-CSF,'"'' whereas TNF-cr inhibits
the proliferative actions of other growth factors such as GCSF, SCF, and CSF-1.'s.2"TNF-cr has been shown to both
inhibit and stimulate erythroid colony growth depending on
the growth factors used, as well as the concentration of TNFa
In the present study, TNF-a invariably
inhibited
BFU-E colony formation of CD34' BM cells induced by
multiple cytokine combinations (Fig 1). In agreement with
previous reports,"." BFU-E colony growth induced by Epo
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
99 1
TNFRECEPTORS IN ERYTHROPOIESIS
alone or Epo in combination with IL-3 was completely inhibited by TNF-a. In addition, and not previously shown, TNFa inhibited BFU-E colony formation induced by SCF + Epo,
IL-3 + SCF + EPO,and IL-9 + EPOby 76%, 70%, and
89%, respectively (Fig 1).
TNF-a inhibited BFU-E colony formation in a concentration-dependent fashion. Fifty percent inhibition of Epo-stimulated colony growth occurred at a TNF-a concentration of
0.02 ng/mL, whereas complete inhibition of erythroid colony-forming ability was observed at 2 ng/mL (Fig 2A). In
comparison, 50% inhibition of SCF + Epo-induced BFU-E
colony growth was observed at a TNF-a concentration of 2
to 20 ng/mL, with a maximum inhibition of 73% occumng
at 200 ng/mL (Fig 2B). Finally, 50% inhibition of SCFinduced granulocyte-macrophage cluster formation was observed at a TNF-a concentration of 0.2 to 2 ng/mL (Fig 2C).
Whereas several previous studies have suggested that
TNF-a can directly modulate the growth of granulocytemacrophage progenitor cell~,"*'~~''
Means et a127328 recently
reported that TNF-a-induced growth inhibition of colonies
derived from human erythroid colony-forming units (CFUE) was indirectly mediated through production of /?-interferon from accessory cells. In contrast, we found that TNFa inhibited the formation of SCF + Epo-induced erythroid
colonies derived from individually plated CD34+ progenitors
in a concentration-dependent fashion, suggesting a direct
action of TNF-a onmore primitive erythroid progenitors
(Fig 3).
Differential effects of TNF-a on IL-3-stimulated granulocyte-macrophage and erythroid progenitors. Because it
has been shown that low, but not high, concentrations of
TNF-a can potently enhance GM-CSF- or IL-3-induced
granulocyte-macrophage colony f~rmation,".'~we next examined the effects of TNF-a on the simultaneous development of CFU-GM and BFU-E colonies in cultures supplemented with IL-3 + Epo. As shown in Fig 4, IL-3 Epo
promoted the growth of 51 2 5 BFU-E and 17 2 2 C m GM colonies per 2 X lo3CD34+ cells plated. Interestingly,
when adding TNF-a at 2 ng/mL, a 2.7-fold increase in the
number of CFU-GM colonies was observed, whereas BFUE colony formation was completely inhibited (Fig 4). At
higher concentration of TNF-a (200 ng/mL), both CFU-GM
and BFU-E colony formation was inhibited. Thus, at low
concentrations, TNF-a has the ability of promoting the
growth of granulocyte-macrophage colonies while concomitantly suppressing the growth of erythroid colonies.
To investigate whether the bifunctional effect of TNF-a
on the growth of CFU-GM and BFU-E progenitors might
be explained by TNF-a preferentially directing primitive
multipotent or bipotent progenitors towards granulocytemacrophage rather than erythroid development or, alternatively, by a differential effect of TNF-a on committed
granulocyte-macrophage and erythroid progenitors, we next
examined the effect of TNF-a on colony formation of
CD34+CD38+CD45RO+and CD34+CD38+CD45RO- cells.
Whereas both these cell populations, lacking CD38 expression, are depleted in primitive progenitor^:^-^^ we found, in
agreement with a previous study," that virtually all BFU-E
(99%) resided in the CD45RO+ subset. In contrast, both
+
A
301
20
T
-
3
m
10-
0-
l+'
0
I
.m2
1
.02
I
.z
T
-
2
20
I
200
TNF-a, nglml
B
3
m
I
0
.W2 .02
1
1
I
1
.2
2
20
200
TNF-a, nglml
C
50
-
E
40
-
3
30
-
20
-
8
U
100-
TNF-a, nglml
Fig 2. Concentration responseof TNF-a-induced inhibition of erythroid progenitor cell proliferation. CD34+ human
cells
BM
were separated as described in the Materials and Methods and cultured
at 2 x
l@
cells/plate in the presence of predetermined optimal connntrations of IAl Epo, (B1 SCF + Epo, or (Cl SCF and increasingconnntrations of TNFa. Cultures werescored for BFU-E colonygrowth (A and
Bl after 14 days and granulocyte-macrophageduster formation IC)
after 10 days of incubation at 37°C and 5% COz in air. Rnutts are
presented as the mean f SEM of three experiments with triplicate
determinations.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
992
RUSTEN AND JACOBSEN
20
T
0
2
11
200
200
TNF-c~,ng/ml
TNFa, ng/ml
Fig 3. Effect of TNF-a on
erythroid colony formation of CD34+ BM
cells in single-cell assay. CD34' cells wereisolated and plated at 1cell
per well as describedin the Materialsand Methods in the
presence of
predetermined optimal concentrations of SCF Epo and different
concentrations of TNF-a as indicated. Erythroid colonies (>40cells)
were assessed(Materialsand Methods) and counted
after 14 days of
incubation at 37°C and 5% CO, in air. In some experiments,individual
colonies were picked to determine that allcolonies formed were in
fact erythroid. Each group consisted of 300 wells. Results are presented as the mean number of
positive wells in four separate experiments: error bars show the SEM.
+
subpopulations contained CFU-GM, although a majority
(60%) were CD45RO-. As shown in Fig 5A, IL-3 + Epo
induced the formation of 17 -+- l CFU-GM colonies and 1.5
? 1 BFU-E colonies of 2,000 CD34+CD38+CD45RO- cells
plated. Interestingly, CFU-GM colony formation of
CD34+CD38+CD45RO- cells increased about fourfold by
adding TNF-a at 2 ng/mL. Thus, the depletion in primitive
progenitors and the removal of progenitors with erythroid
T
1
*
U
2
0
200
2
TNF
U
-,
nglml
Fig 5. Effects of TNF-a on IL-3-stimulated ( 0 )CFU-GM and (W)
BFU-E colony formation by (AI CD34+CD38+CD&RO- cells and (B)
CD34+CD38+CD&RO+ cells. Progenitors were isolated and plated in
methylcellulose as described in the Materials and Methods at 2 x
lo3cellslplate. Individual cultures were supplemented
with predetermined optimal concentrations of 11-3 + Epo anddifferent concentrations of TNF-a as indicated. Cultures were scoredfor CFU-GM and
BFU-E colony growth after 14 days ofincubation at 37°C and 5% CO,
in air. Results are presented as the mean number of colonies per
2 x 10' cells from three independent experiments with duplicate
determinations; error bars show the SEM. *No BFU-E colony formation.
60
0
1401
200
TNF-U, ng/ml
Fig 4. Effects of TNFa on IL-3-stimulated ( 0 )CFU-GM and (.l
BFU-E colony formation by human BM progenitor cells. CD84+ cells
were isolated and plated in methylcellulose as described
in the Matexa
l cellslplate. Individual cultures were
rials and Methods at 2 O
supplemented with predetermined optimal concentrations of IL-3 +
Epoand different concentrations of TNF-a as indicated. Cultures
were scored for CN-GM and BFU-E colony growth after 14 days of
incubation at 37°C and 5% CO, in air. Results are presented as the
mean number of colonies per 2 x 10s cells from three independent
experiments with triplicate determinations; error bars
show the SEM.
*No BFU-E colony formation.
potential from the test cell population did not impair the
ability of TNF-a to enhance IL-3-stimulated CFU-GM colony formation. Using CD34+CD38*CD45RO+cells, IL-3 +
Epo stimulated the growth of 23 ? I CFU-GM and 114
? 13 BFU-E colonies of 2,000 cells plated (Fig 5B). The
supplementation of TNF-a at 2 ng/mL almost completely
blocked BFU-E colony formation, whereas CFU-GM colony
numbers increased 2.5-fold. Thus, enriching progenitors capable of erythroid colony formation did not further enhance
the ability of TNF-a to stimulate CFU-GM colony formation.
Role ofp55 andp75TNFRs in TNF-a-induced inhibition
of erythropoiesis. In a previous study, we have shown that
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
993
TNF RECEPTORS IN ERYTHROPOIESIS
*
B
*
100
growth factors, Epo promoted the formation of 18 5 2 BFUE colonies per 2 X lo3 CD34+ cells plated, and TNF-a as
well as TNFR-p55 agonists (TNF-a mutant or anti-TNFRp55 antibody htr-9) completely inhibited Epo-induced BFUE colony formation (Fig 6A). Interestingly, TNFR-p75 agonists (TNF-a mutant or anti-TNFR-p75 antibody) could
also partially inhibit Epo-induced BFU-E colony growth
(41%; P < .05). Whereas TNF-a! inhibited SCF + Epostimulated BFU-E colony growth by 86%, the inhibition
induced by the TNFR-p55 and TNFR-p75 selective agonists
was 66% (P< .05) and 20%,respectively (Fig 6B). Combining the two TNF-a mutants had little additional inhibitory
effect as compared with the TNFR-p55-specific mutant
alone (data not shown). In agreement with aprevious study,"
the p55 TNF-a mutant, but not the TNF-a mutant specific
for TNFR-p75 nor the agonistic anti-TNFR-p75 antibody,
stimulated IL-3- and inhibited G-CSF-induced colony formation (data not shown), confirming the specificity of these
reagents. Thus, TNF-a-induced inhibition of BFU-E colony
formation from CD34+ progenitors is predominantly mediated through TNFR-p55, but might also involve the TNFRp75.
DISCUSSION
Fig 6. Relative roleof p55 and p75 TNFRs
in TNF-a-induced inhibition of BFU-E colony formation. CD34+ cells were isolated and plated
in me?hylcellulose as describd in the Materials and Methodsat 2 x
lo'cellslplate in the presence of predetermined optimal concentrations of (A) Epoor IBI SCF + Epo. TNF-a (200 nglmL), TNFR-p55
agonists (TNFa mutant at 200 nglmL or htr-9 at 10 pglmLI, or TNFRp75 agonistsm F a mutant at 2 pglmL or paTNFFkp75at 2 pglmLI
were added as indicated. Results are presented as
the mean number
of colonies per2 x l@
cdls from six independent experiments with
tripliite determinations, including three experiments using TNFa
mutants andthree experimentswithanti-TNFR antibodies;error bars
show the SEM. *No colony formation.
TNFR-p55 exclusively mediates the potentiating effect of
TNF-a on GM-CSF- or IL-3-induced proliferation of
CD34+ progenitor cells as well as inhibition of G-CSFstimulated colony formation, whereas the TNFR-p75 is involved in mediating inhibitory effects on the proliferation
of primitive high proliferative potential colony-forming
cells." In the present study, we investigated the relative roles
of the two TNFRs in mediating the inhibitory effects of
TNF-a on BFU-E colony formation. Similar results were
obtained with TNF-a mutants and anti-TNFR antibodies
with selective agonistic activity on either of the two receptor
types (Fig 6). In the absence of other exogeneously added
TNF-a has been demonstrated to signal both inhibition
and stimulation of hematopoietic progenitor cells."Ls*20
Specifically, TNF-a has been shown to potently inhibit in vitro
erythropoiesis stimulated by Epo alone or in combination
with IL-3.".I3 We demonstrate here for the first time that
TNF-a also potently inhibits BFU-E colony formation stimulated by both SCF + Epo and IL-9 + Epo. Of particular
interest is the finding that TNF-a inhibits SCF-stimulated
erythroid colony formation, because SCF has been shown
to be maybe the most potent stimulator of erythrop~iesis.".~~
The EDJo of TNF-a-induced inhibition of SCF + Epostimulated BFU-E colony formation was 1,000-fold higher
than that observed for TNF-a-induced inhibition of colony
formation stimulated by Epo alone and = 10-fold higher than
that observed for SCF-induced cluster formation. This observation is of particular interest because it might suggest that
combinations of stimulatory growth factors can cooperate to
overcome the growth inhibitory effects of TNF-a. Thus,
growth factors such as SCF might be used to reverse the
anemia observed after TNF-a administration and in chronic
disea~es.''~*~~
In a similar fashion, previous studies have
shown that transforming growth factor-P-induced inhibition
of progenitor cell growth can be abrogated either by increasing the concentration of stimulatory growth factorsm or by
supplementing additional growth factors:' Alternatively, it
is possible that the presumably more primitive subset of
progenitors recruited by SCF + Epo is less sensitive to TNFCY than the subset of those responding to Epo alone.
The effects of TNF-a can be mediated directly on target
cells1zp16
or indirectly by stimulating accessory cells to cytokine prod~ction.~"'~
Whereas we and others have found direct effects of TNF-a on committed granulocyte-macrophage
BM progenitor cell^,'^^'^ recent studies by Means et a127*28
have suggested that TNF-a-induced inhibition of erythroid
colony growth was indirect through stimulation of &inter-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
994
feron production from accessory cells. In contrast, the results
of single-cell cloning experiments presented here suggest
that TNF-a-induced inhibition of erythroid colony formation can be directly mediated on the progenitor cells. The
divergent findings in the studies of Means et alz7***
and in
our study could be due to studying different erythroid progenitors, because our results were obtained assessing TNFa-induced modulation of BFU-E colony formation stimulated by multiple cytokine combinations, whereas Means et
a1 evaluated TNF-a -induced inhibition of colony formation
derived from more mature CFU-E progenitors stimulated by
Epo alone. It is possible that TNFR expression and function
varies among these two progenitor cell types. Alternatively,
TNFR expression might differ on progenitors of the same
type according to the the conditions under which they have
been generated.
It is well established that TNF-a can inhibit or stimulate
the proliferation of hematopoietic progenitor cells, depending on the growth factor(s) it interacts with as well as
the concentration of TNF-a in culture."-15 The potent TNFa-induced enhancement of CFU-GM colony growth and
inhibition of erythroid colony formation stimulated by IL-3
+ Epo is of particular interest, because it demonstrates that
one concentration of TNF-a can simultaneously and bidirectionally modulate IL-3-dependent growth of granulocytemacrophage (stimulation) and erythroid colonies (inhibition). The present study suggests that this can be explained
by a differential effect of TNF-a on committed granulocytemacrophage and erythroid progenitors and not by TNF-a
switching more primitive multipotent or bipotent progenitors
from erythroid towards granulocyte-macrophage development. This theory was supported by TNF-a retaining its
ability to enhance IL-3-induced CFU-GM colony growth
when the test cell population was depleted in primitive and
erythroid progenitors, as well as by the fact that TNF-ainduced potentiation of L-3
-dependent CFU-GM colony
formation was not further enhanced using a test cell population highly enriched in progenitors with erythroid potential.
Recent studies from our laboratory suggest that both
TNFR-p55 and TNFR-p75 are involved in mediating TNFa-induced inhibition of primitive hematopoietic progenitor
cells requiring multiple cytokines to pr01iferate.I~In contrast,
the TNFR-p55 excusively mediates stimulatory effects on
more mature GM-CSF- or IL-3-responsive granulocytemacrophage progenitor cells, as well as potent inhibition
of G-CSF-induced proliferation. The present study extends
these findings to show that TNF-a-induced inhibition of
BFU-E colony formation in response to multiple cytokine
combinations is mediated predominantly through TNFRp55. However, whereas signaling through TNFR-p75 had no
effect on the proliferation of committed granulocyte-macrophage progenitor cells,I5 weshow here that this receptor type
can mediate inhibition of committed erythroid progenitor
cell growth. Although a small inhibition of SCF + Epoinduced BFU-E colony formation was observed, the TNFRp75-mediated inhibition was more obvious on progenitors
responding to Epo alone, indicating a predominant effect on
more mature stages of erythroid progenitors. These findings
might have several important clinical implications. First,
RUSTEN AND JACOBSEN
TNFR-p55--specific mutant TNF-a has been shown to have
less proinflammatory (toxic) effects than wild-type TNF-a;'
but as potent cytotoxic (antitumor) effects, and is thus being
explored for the possible use as an antitumor agent. However, the present results suggest that the TNF-a mutant specific for TNFR-p55 is as potent as wild-type TNF-a in inhibiting erythropoiesis, andmight
therefore induce severe
anemia. In contrast, administration of TNF-a mutants with
selectivity for TNFR-p75 might be associated with little or
no anemia. Second, antagonistic antibodies to TNFR-p55
could potentially be used to reverse the anemia observed in
chronic diseases.
It has been shown that some TNFR-p55-mediated responses are much more efficiently elicited by wild-type
TNF-a than TNFR-p55--selective TNF-a mutants, suggesting an accessory role of TNFR-p75 in TNFR-p55-signaled event^.^*.^^ It is of interest that the data presented here,
as well as in previous studies,l5." suggest that this does not
appear to be the case for TNFR-p55-mediated regulation of
human hematopoietic progenitor cell growth.
In conclusion, we have demonstrated that TNF-a potently
and directly inhibits the in vitro growth of committed erythroid progenitor cells in response to multiple cytokine combinations, and that TNF-a-induced inhibition of BFU-E colony formation ismainly
mediated through TNFR-p55,
although TNFR-p75 -mediated inhibition could be observed
on progenitors responsive to Epo alone. Ongoing studies on
TNF receptor knock-out mice46 willhelp to further elucidate
the role of TNF-a and its receptors in erythropoiesis
ACKNOWLEDGMENT
Theauthorsaregrateful to Ole P. Veiby for the cell sorting, to
Dr ErlendB. Smeland for support during these
studies, and to Cecilie
Okkenhaug and Eli Lien for expert technical
assistance.
REFERENCES
1. SugarmanBJ, Agganval BB, HassPE,Figari IS, Palladino
MA, Shepard H M : Recombinanthumantumor necrosis factor-cy:
Effects on proliferation of normalandtransformed cells in vitro.
Science 230:943, 1985
2. Vilcek J, Palombella VJ, Henriksen-deStefano D, Swenson C,
FeinmanR,Hirai M, Tsujimoto M:Fibroblastgrowthenhancing
activity of tumor necrosis factor andits relationship to other polypeptide growth factors. J Exp Med 163:632, 1986
3. Nawroth PP, Stern DM: Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 163:740, 1986
4. ShalabyMR, Agganval BB, Bnderknecht E,SvederskyLP,
Finkle BS, Palladino MA: Activation of human polymorphonuclear
neutrophilfunctionsbyinterferon-yandtumor
necrosis factors. J
Immunol 135:2069, 1985
5. Kelker HC, Oppenheim JD, Stone-Wolff D, Henriksen-deStefano D, Agganval BB, Stevenson HC, Vilcek J: Characterization of
human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin. Int J Cancer 3669, I985
6. Cuturi MC, MurphyM, Costa-Giomi MP, Weinmann R, Perussia B, Trinchieri G: Independent regulation of tumor necrosis factor
andlympbotoxinproductionbyhumanperipheralbloodlymphocytes. J ExpMed 165:1581, 1987
7. Degliantoni G, Murphy M, Kobayashi M, Francis MK, Perussia B, Trinchieri G: Natural killer (NK) cell-derived hematopoietic
colony-inhibitingactivity and NKcytotoxic factor: Relationship with
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
TNF RECEPTORS IN ERYTHROPOIESIS
tumor necrosis factor and synergism with immune interferon. J Exp
Med162:1512, 1985
8. Broxmeyer HE, Williams DE, Lu L, Cooper S , Anderson SL,
Beyer CS, Hoffman R, Rubin BY: The suppressive influences of
human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: Synergism of tumor necrosis factor and interferon-y. J Immunol 136:4487,
1986
9. Murase T, Hotta T, Saito H, Ohno R: Effect of recombinant
human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells.
Blood 69:467, 1987
IO. Murphy M, Perussia B, Trinchieri G: Effects of recombinant
tumor necrosis factor, lymphotoxin, and immune interferon on proliferation and differentiation of enriched hematopoietic precursor
cells. Exp Hematol 16:131, 1988
11. Backx B, Broeders L, Bot FJ, Lswenberg B: Positive and
negative effects of tumor necrosis factor on colony growth from
highly purified normal marrow progenitors. Leukemia 5:66, 1991
12. Caux C, Saeland S , Favre C, Duvert V, Mannoni P, Banchereau J: Tumor necrosis factor-alpha strongly potentiates interleukin3 and granulocyte-macrophage colony-stimulating factor-induced
proliferation of human CD34+ hematopoietic progenitor cells. Blood
75:2292, 1990
13. Caux C, Favre C, Saeland S , Duvert V, Durand I, Mannoni P,
Banchereau J: Potentiation of early hematopoiesis by tumor necrosis
factor-a is followed by inhibition of granulopoietic differentiation
and proliferation. Blood 78:635, 1991
14. Caux C, Durand I, Moreau I, Duvert V, Saeland S , Banchereau J: Tumor necrosis factor a cooperates with interleukin 3 in the
recruitment of a primitive subset of human CD34+ progenitors. J
Exp Med 177:1815, 1993
15. Rusten LS, Jacobsen FW, Lesslauer W, Loetscher H, Smeland
EB, Jacobsen SEW: Bifunctional effects of TNF-a on the growth
of mature and primitive human hematopoietic progenitor cells:
Involvement of p55 and p75 TNF receptors. Blood 83:3152, 1994
16. Jacobsen SEW, Ruscetti FW, Dubois CM, Keller JR: Tumor
necrosis factor a directly and indirectly regulates hematopoietic progenitor cell proliferation: Role of colony-stimulating factor receptor
modulation. J Exp Med 175:1759, 1992
17. Munker R, Gasson J, Ogawa M, Koeffler HP: Recombinant
human TNF induces production of granulocyte-monocyte colonystimulating factor. Nature 323:79, 1986
18. Oster W, Lindemann A, Horn S, Mertelsmann R, Hemnann
F Tumor necrosis factor (TNF)-a but not TNF-p induces secretion
of colony stimulating factor for macrophages (CSF-1) by human
monocytes. Blood 70:1700, 1987
19. Koeffler HP, Gasson J, Ranyard J, Souza L, Shepard M,
Munker R: Recombinant human TNF-a stimulates production of
granulocyte colony-stimulating factor. Blood 7055, 1987
20. Rusten LS, Smeland EB, Jacobsen FW, Lien E, Lesslauer W,
Loetscher H, Dubois CM, Jacobsen SEW: TNF-a inhibits stem cell
factor-induced proliferation of human bone marrow progenitor cells
in vitro: Role of p55 and p75 TNF receptors. J Clin Invest 94:165,
1994
21. Brach MA, Biihring HJ, Gruss HJ, Ashman LK, Ludwig WD,
Mertelsmann RH, Hemnann F Functional expression of c-kit by
acute myelogenous leukemia blasts is enhanced by tumor necrosis
factor-a through posttranscriptional mRNA stabilization by a labile
protein. Blood 801224, 1992
22. Branch DR. Turner AR, Guilbert LJ: Synergistic stimulation
of macrophage proliferation by the monokines tumor necrosis factora and colony-stimulating factor-l. Blood 73:307, 1989
23. Shieh JH, Peterson RHF, Warren DJ, Moore MAS: Modula-
995
tionof colony-stimulating factor-l receptors on macrophages by
tumor necrosis factor. J Immunol 143:2534, 1989
24. Elbaz 0, Budel LM, Hoogerbrugge H, Touw P,Delwel R,
Mahmoud LA, Lswenberg B: Tumor necrosis factor regulates the
expression of granulocyte-macrophage colony-stimulating factor and
interleukin-3 receptors on human acute myeloid leukemia cells.
Blood 77:989, 1991
25. Shieh JH, Peterson RHF, Moore MAS: Modulation of granulocyte colony-stimulating factor receptors on murine peritoneal exudate macrophages by tumor necrosis factor-a. J Immunol 146:2648,
1991
26. Elbaz 0, Budel LM, Hoogerbrugge H, Touw IP, Delwel R,
Mahmoud LA, Lrawenberg B: Tumor necrosis factor downregulates
granulocyte-colony-stimulatingfactor receptor expression on human
acute myeloid leukemia cells and granulocytes. J Clin Invest 87:838,
1991
27. Means RT, Dessypris EN, Krantz SB: Inhibition of human
colony-forming-unit erythroid by tumor necrosis factor requires accessory cells. J Clin Invest 86:538, 1990
28. Means RT, Krantz SB: Inhibition of human erythroid colonyforming units by tumor necrosis factor requires beta interferon. J
Clin Invest 91:416, 1993
29. Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GHW,
Gatanaga T, Granger CA, Lentz R, Raab H, Kohr WJ, Goeddel DV:
Molecular cloning and expression of a receptor for human tumor
necrosis factor. Cell 61:361, 1990
30. Loetscher H, Pan YCE, Lahm HW, Gentz R, Brockhaus M,
Tabuchi H, Lesslauer W Molecular cloning and expression of the
human 55 kd tumor necrosis factor receptor. Cell 61:351, 1990
31. Heller RA, Song K, Onasch MA, Fischer WH, Chang D,
Ringold GM: Complementary DNA cloning of a receptor for tumor
necrosis factor and demonstration of a shed form of the receptor.
Proc Natl Acad Sci USA 87:6151, 1990
32. Dembic Z, Loetscher H, Gubler U, Pan YC, Lahm H W , Gentz
R, Brockhaus M. Lesslauer W: Two human TNF receptors have
similar extracellular, but distinct intracellular, domain sequences.
Cytokine 2:231, 1990
33. Smith CA, Davis T, Anderson D, Solam L, Beckmann MP,
Jerzy R, Dower SK, Cosman D, Goodwin RC: A receptor for tumor
necrosis factor defines an unusual family of cellular and viral proteins. Science 248:1019, 1990
34. Engelmann H, Holtmann H, Brakebusch C, Avni YS, Sarov
I, Nophar Y,Hadas E, Leitner 0, Wallach D: Antibodies to a soluble
form of a tumor necrosis factor (TNF) receptor have TNF-like activity. J Biol Chem 265:14497, 1990
35. Espevik T, Brockhaus M, Loetscher H, Nonstad U, Shalaby
R: Characterization of binding and biological effects of monoclonal
antibodies against a human tumor necrosis factor receptor. J Exp
Med171:415, 1990
36. Shalaby MR, Sundan A, Loetscher H, Brockhaus M, Lesslauer W, Espevik T: Binding and regulation of cellular functions
by monoclonal antibodies against human tumor necrosis factor receptors. J Exp Med 172:1517, 1990
37. Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino
MA, Goeddel CV: The two different receptors for tumor necrosis
factor mediate distinct cellular responses. Proc Natl Acad Sci USA
88:9292, 1991
38. Tartaglia LA, Goeddel DA, Reynolds C, Figari IS, Weber
RF, Fendly BM, Palladino MA: Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor
receptor. J Immunol 151:4637, 1993
39. Heller RA, Song K, Fan N. Chang DJ: The p70 tumor necrosis
factor receptor mediates cytotoxicity. Cell 70:47, 1992
40. Bigda J, Beletsky I, Brakebusch C, Varfolomeev Y, Engelmann H, Bigda J, Holtmann H, Wallach D: Dual role of the p75
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
996
tumor necrosis factor (TNF) receptor in TNF cytotoxicity. J Exp
Med 180:445, 1994
41. Jacobsen FW, Rothe M, Rusten LS, Goeddel DV, Smeland
EB, Slsrdal L, Jacobsen SEW: Novel role ofthe 75-kDa tumor
necrosis factor (TNF) receptor: inhibition of early hematopoiesis.
Proc Natl Acad Sci USA 91:10695, 1994
42. Means RT, Krantz SB: Progress in understanding the pathogenesis of the anemia of chronic disease. Blood 80:1639, 1992
43. Tracey KJ, Beutler B, Lowry SF, Menyweather 3, Wolpe S,
Milsark IW, Hariri RJ, Fahey TJ 111, Zentella A, Albert JD, Shires
GT, Cerami A: Shock and tissue injury induced by recombinant
human cachectin. Science 234:470, 1986
44. Van Ostade X, Vandenabeele P, Everaerdt B, Loetscher H,
Gentz R, Brockhaus M, Lesslauer W, Tavernier J, Brouckaert P,
Fiers W: Human TNF mutants with selective activity on the p55
receptor. Nature 361:266, 1993
45. Balkwill F Improving the formula. Nature 361:206, 1993
46. Rothe J, Lesslauer W, Lotscher H, Lang Y, Koebel P, Kontgen F, Althage A, Zinkernagel R, Steinmetz M, Bluethmann H: Mice
lacking the tumor necrosis factor receptor 1 are resistant to TNFmediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364:798, 1993
47. Loetscher H, Stueber D, Banner D, Mackay F, Lesslauer W:
Human tumor necrosis factor (U (TNFa) mutants with exclusive
specificity for the 55-kDa or 75-kDa TNF receptors. J Biol Chem
268:26350,1993
48. Barbara JAJ, Smith WB, Gamble JR, Van Ostade X, Vande' nabeele P, Tavernier J, Fiers W, Vadas MA, Lopez AF: Dissociation
of TNF-(Ucytotoxic and proinflammatory activities by p55 receptorand p75 receptor-selective TNF-a mutants. EMBO J 135343, 1994
49. Gehr G, Gentz R, Brockhaus M, Loetscher H, Lesslauer W:
Both tumor necrosis factor receptor types mediate proliferative signals in human mononuclear cell activation. J Immunol 149:911,
1992
50. Brockhaus M, Schoenfeld HJ, Schlaeger El, Hunziker W,
Lesslauer W, Loetscher H: Identification of two types of tumor
necrosis factor receptors on human cell lines by monoclonal antibodies. Proc Natl Acad Sci USA 87:3127, 1990
5 1. Smeland EB, Funderud S, Kvalheim G, Gaudernack G, Rasmussen A-M, Rusten L, Wang MY, Tindle RW, Blomhoff HK,
Egeland T: Isolation and characterization of human hematopoietic
RUSTEN AND JACOBSEN
progenitor cells: An effective method for positive selection of CD34'
cells. Leukemia 65345, 1992
52. Lansdorp PM, Sutherland HJ, Eaves CJ: Selective expression
of CD45 isofoms on functional subpopulations of CD34' hemopoietic cells from human bone marrow. J Exp Med 172:363, 1990
53. Terstappen LWMM, Huang S, Safford M, Lansdorp PM, Loken MR: Sequential generations of hematopoietic colonies derived
from single nonlineage-committed CD34+CD38- progenitor cells.
Blood 77:1218, 1991
Lymphoid and myeloid differ54. Huang S, Terstappen LW":
entiation of single human CD34', HLA-DR+, CD38- hematopoietic
stem cells. Blood 83:1515, 1994
55. Rusten LS, Jacobsen SEW, Kaalhus 0, Veiby OP, Funderud
S, Smeland EB: Functional differences between CD38- and DRsubfractions of CD34+ bone marrow cells. Blood 84:1473, 1994
56. Gregory CJ, Eaves AC: Human marrow cells capable of erythropoietic differentiation in vitro: Definition of three erythroid colony
responses. Blood 492355, 1977
57. Broxmeyer HE, Cooper S, LuL, Hangoc G, AndersonD,
Cosman D, Lyman SD, Williams DE: Effect of murine mast cell
growth factor (c-kit proto-oncogene ligand) on colony formation by
human marrow hematopoietic progenitor cells. Blood 77:2142, 1991
58. McNiece IK, Langley KE, Zsebo KE. Recombinant human
stem cell factor synergises with GM-CSF, G-CSF, L 3 and Epo to
stimulate human progenitor cells of the myeloid and erythroid lineages. Exp Hematol 19:226, 1991
59. Johnson RA, Waddelow TA, Caro J, Oliff A, Roodman GD:
Chronic exposure to tumor necrosis factor in vivo preferentially
inhibits erythropoiesis in nude mice. Blood 74:130, 1989
60. Cashman JD, Eaves AC, Eaves CJ: Granulocyte-macrophage
colony-stimulating factor modulation of the inhibitory effect of
transforming growth factor-p on normal and leukemic human hematopoietic progenitor cells. Leukemia 8:886, 1992
61. Jacobsen SEW, Ruscetti FW, Ortiz M, Gooya JM, Keller JR:
The growth response of Lin-Thy-l+ hematopoietic progenitors to
cytokines is determined by the balance between synergy of multiple
stimulators and negative cooperation of multiple inhibitors. Exp
Hematol 22:985, 1994
62. Tartaglia LA, Pennica D, Goeddel DV: Ligand passing: The
75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 268:18542, 1993
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 85: 989-996
Tumor necrosis factor (TNF)-alpha directly inhibits human
erythropoiesis in vitro: role of p55 and p75 TNF receptors
LS Rusten and SE Jacobsen
Updated information and services can be found at:
http://www.bloodjournal.org/content/85/4/989.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.