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1995 86: 2559-2567
AM Gewirtz, J Zhang, J Ratajczak, M Ratajczak, KS Park, C Li, Z Yan and M Poncz
Chemokine regulation of human megakaryocytopoiesis
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Chemokine Regulation
of
Human Megakaryocytopoiesis
By
Alan
M.
Gewirtz, Jin Zhang, Janina Ratajczak, Mariusz Ratajczak, Kwang Sook Park,
Changqing Li, Zhanqing Yan, and Mortirner Poncz
We have previously shown that platelet factor 4 (PF4). a
platelet-specific CXC chemokine, can directly and specifically
inhibit
human megakaryocyte colony formation. We there-
fore hypothesized that PF4 might function as a negative au-
tocrine regulator of megakaryocytopoiesis. Herein we pre-
sent additional studies characterizing the inhibitory effect
of CXC chemokines on human megakaryocyte development.
We
first
corroborated our initial studies by showing that
recombinant human (rH1 PF4, like the native protein,
inhib-
ited megakaryocytopoiesis. We then examined the
inhibi-
tory properties of other CXC family members. Neutrophil
activating peptide-2 (NAP-2). a naturally occurring N-termi-
nally cleaved PTG peptide, was found to inhibit megakaryo-
cytopoiesis
with
two
to three orders of magnitude greater
potency than PF4. Structure function studies showed that an
N-terminal mutation, which eliminated NAP-2's neutrophil
activating properties (NAP-2mA), also abrogated its ability
to
inhibit
megakaryocyte development. Further investiga-
tions of this type demonstrated that a chimeric PF4 protein
(AELR/PF4)
in
which PF4's N-terminus was replaced with the
first four amino acids of NAP-P was also a potent inhibitor of
UMAN MEGAKARYOCYTOPOIESIS and platelet
production are complex processes whose regulation
remains incompletely understood. The identification and
cloning of the c-mpl ligand, now known as thrombopoietin
(TPO) or MGDR, represents
an
important advance in this
area because this protein appears to be the major regulator
of megakaryocyte development. MGDR has been shown,
for example, to increase the number, size, and ploidy of
megakaryocytes, and in vivo administration markedly in-
creases platelet count in recipient anirnal~."~ Nevertheless,
MGDR is clearly not the sole regulator of the megakaryo-
cytelplatelet axis, as innumerable cytokine experiments5"'
and knockout experiments of the MGDR receptor, c-Mpl,"
have shown.
Besides positive effectors, it is possible that megakaryocy-
topoiesis is regulated by inhibitory influences as well. In
this regard, it has been observed that megakaryocyte colony
growth is inferior in serum than in platelet-poor plasma sug-
gesting that a platelet released product inhibits megakaryo-
cyte gro~th.'~"~ Megakaryocytes
also
appear to release prod-
ucts that inhibit their own development. We, and others,
have shown that one of the platelet-specific a-granule chem-
okines, platelet factor 4 (PF4), can inhibit megakaryocyte
colony formation in a lineage-specific fashion.I6"' Inhibition
is manifested by a decrease in the number and size of mega-
karyocyte colonies. In our hands,16 the related platelet-spe-
cific a-granule chemokine, PTG, does not exert similar ef-
fects on cultured cells, though conflicting findings have been
reported by others." In aggregate, these studies suggest that
megakaryocytopoiesis and thrombopoiesis may be con-
trolled to an as yet undetermined extent by a negative auto-
crine growth loop.
One caveat in attributing a physiologic role to PF4 in
the regulation of human megakaryocyte development is the
amount of material required to observe the negative growth
effect. Though some markers of megakaryocyte maturation,
H
Blood,
Vol
86,
No
7 (October
l),
1995:
pp
2559-2567
megakaryocytopoiesis. lnterleukin (IL)-8, another CXC
chemokine, and three CC chemokines (macrophage
inhibi-
tory protein-l& [MIP-la], MIP-1/3, and C101 also specifically
inhibited megakaryocyte colony formation at NAP-2 equiva-
lent doses. CXC and CC chemokine inhibition was additive
suggesting that the effects might be mediated through a
common pathway. The inhibitory effects of NAP-2 and MIP-
la
could not be overcome by adding physiologically relevant
amounts of recombinant human megakaryocyte growth and
development factor (MGDR) (50 ng/mL) to the cultures.
Us-
ing Northern blot and reverse transcriptase-polymerase
chain reaction (RT-PCR) based analyses, we documented
mRNA expression of IL-8 receptor isoforms
a
and
/3
in
total
platelet RNA and
in
normal human megakaryocytes, respec-
tively. Based on these results, we hypothesize that chemo-
kines play a physiologic role
in
regulating megakaryocyto-
poiesis. Because chemokines are elaborated by ancillary
marrow cells, both autocrine and paracrine growth control
is suggested, the
effects
of which might be exerted,
in
part,
through
a
and
/3
IL-8 receptors.
0
7995
by
The American Society
of
Hematology.
for example factor
V
mRNA expression, could be inhibited
by nanogram quantities of PF4, colony inhibition occurs in
the range of
-25
pg/mL.I6 Though such concentrations are
routinely observed in serum, whether marrow concentrations
of this magnitude can really be achieved in vivo is debatable.
Accordingly, we entertained the possibility that other mem-
bers of the chemokine family to which PF4 and PTG be-
longed might possess similar, or perhaps even more potent,
inhibitory activity against the megakaryocyte lineage.I9-*'
Chemokines are
70
to
100
amino acids long and contain
four cysteine resid~es.l~-~~ The family has diverged into two
main subgroups. PF4 and PTG are members of the
CXC
or
a
chemokine family subgroup. In this group the first two of
the four conserved cysteine residues are separated from each
other by one additional amino acid residue. This subfamily
also includes interleukin-8
(IL-8),
a potent neutrophil acti-
vating agent. PF4 and
PTG
are poor neutrophil activators,
but a cleavage product of PTG, neutrophil activating peptide-
From the Departments of Pathology, Medicine and Pediatrics,
University of Pennsylvania School
of
Medicine, Philadelphia; The
Children's Hospital of Philadelphia, Philadelphia, PA; and the De-
partment of Microbiology, Korea University School of Medicine,
Seoul, Korea.
Submitted January 9, 1995; accepted June 2, 1995.
Supported by Grants No. CA36896 to A.M.G. and HL.37419 to
M.P. from the National Institutes of Health and the Tobacco Re-
search Council Grant
No.
3152 to M.P.
Address reprint requests
to
Mortimer Poncz, MD, The Children
'S
Hospital of Philadelphia, 34th St and Civic Center Blvd, Philadel-
phia, PA 19104.
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
I8
U.S.C. section 1734
solely
to
indicate this fact.
0
1995 by The American Society of Hematology.
0006-4971~.5/8607-0019$3.00/0
2559
For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom
2560
GEWIRTZ
ET
AL
2
(NAP-2),
is
a
potent neutrophil activat~r.~~”~ Of note, two
different
IL-8
receptors
(IL-8R)
are
expressed by neutro-
phils. One is designated the
a
or
type
I
IL-8R
and binds
IL-8
efficiently, but
NAP-2
poorly.
A
second
/?
or type
2
IL-8R
has also been described, which binds
IL-8
well, and
NAP-2
with moderate affinity.20.26,27
The
CC or
p
chemo-
kines, in which the first
two
cysteine residues
are
immedi-
ately adjacent
to
each
other,
represent the other branch
of
this family. CC chemokines such
as
macrophage inhibitory
protein
(MIP)-la**
appear to be important
monocyte
stimu-
lating agents and
may
also selectively suppress human
mega-
karyocyte development.
A
CC chemokine receptor has been
defined.*’
The
PF4
and
PTG
studies described above suggested that
neutrophil activating potency and megakaryocyte colony in-
hibitory activity paralleled each other. To test this hypothe-
sis,
we
examined
the effect of the neutrophil activator
NAP-
2
on human megakaryocyte development in vitro.
We
also
sought
to
determine
the
megakaryocyte colony inhibitory
activity of other
members
of this chemokine family. Herein,
we present data showing that
a
number
of
CXC
and
CC
chemokines
are
potent inhibitors of human megakaryocyto-
poiesis and that megakaryocytes
express
a
number of chemo-
kine receptors. These data suggest that chemokines, perhaps
elaborated by
ancillary
cells in the marrow microenviron-
ment,
may
play
a
physiologically significant role in regulat-
ing human megakaryocyte development
by
both
autoctine
and paracrine mechanisms.
MATERIALS
AND
METHODS
Cells
Light-density bone marrow mononuclear cells (MNC) were ob-
tained from normal remunerated consenting donors and depleted of
adherent cells and T lymphocytes (A-T-MNC) as described.3o
CD34’ were enriched from the A-T-MNC population by incubation
with anti-human progenitor cell antigen (HPCA)-1 murine mono-
clonal antibodies (Becton Dickinson, San Jose, CA) and subsequent
immunoselection of antibody-labeled cells with magnetic beads ac-
cording to the manufacturer’s protocol (Dynal,
Oslo,
Norway) as
described.” Purity of CD34’ cells selected in this manner exceeded
85%.
Hematopoietic Colony Assays
All
assays were performed with either peripheral blood or bone
marrow obtained from normal consenting volunteers. Hematopoietic
colonies were grown and identified
as
previously In
brief, colony assays were performed with either unseparated light-
density marrow nuclear cells (MNC)
or
with MNC depleted of
monocyte-macrophages and T-lymphocytes as previously de-
scribed.” Final plated cell concentrations were
2
X
10’lmL. Cultures
were supplemented with
30%
vollvol of aplastic anemia serum, ex-
cept when supplemented with recombinant human MGDF (gener-
ously provided by Dr Pamela Hunt, Amgen Corp, Thousand Oaks,
CA).4 Megakaryocyte and granulocyte colonies were supplemented
with
rH
IL-3
(-29
U/mL) and rH granulocyte-macrophage colony-
stimulating factor (GM-CSF) (-5 ng/mL) (Genetics Institute, Cam-
bridge, MA), except for the megakaryocyte studies that tested MGDF
effect. Erythroid colonies were supplemented with recombinant
erythropoietin (5 U/mL) (Amgen Corp). Recombinant human chem-
okines were added just before plating.
Megakaryocyte colonies were enumerated after
12
days in culture
by indirect immunofluorescence using either a highly-specific rabbit
antihuman platelet membrane glycoprotein antiserum’h~3”.31 or :I
monoclonal anti-GPIIbflIIa complex antibody
A2AY.”
Binding
of
the probe antibody was detected with a species appropriate Ruores-
cein-conjugated secondary antibody (Meloy, Springfield, VA). A
cluster of three
or
more intensely fluorescent cells were defined as
one colony. Plates were read by two separate individuals, including
one blinded to plate designation. Results were in agreement between
both readers within
5%
to 10%. Results
are
reported
as
the mean
L
standard error (SE)
of
colonies enumerated.
Colony forming units-erythroid (CFU-E) were cultured
for
7
days
and then enumerated after staining with
I
%
benzidine and hematoxy-
lin as previously described.’” Colony-forming unit granulocyte-mac-
rophage (CFU-GM) were cultured and identified as previously de-
scribed.3”
In
Vitro Synthesis
of
Recombinant Human Chemokines
The construction of the T,-promoter expression vectors for PF4,
NAP-2, AELRPF4, in which the N-terminal amino acid sequence
of
PF4 preceding the first cysteine residue is replaced with the amino
acids A-E-L-R, and NAP-2“2’A in which the second amino acid
residue of NAP-2 is mutated from glutamic acid to alanine has
been previously described.” These vectors were used to express
recombinant proteins in
Escherichia coli
BL21(DE3)pLys
S
(Nova-
gen. Madison,
WI),
which were allowed
to
grow to an optical density
(OD),, of -0.9, which was followed by an additional
3
hours of
growth in
1
mmol/L
isopropyl-thiogalactopyranoside
that induced
recombinant protein expression.
The recombinant proteins were processed as previously
de-
~cribed.’~.~~ Bacteria were centrifuged at
3,OOO
rpm
in
a Sorvall
GSA
rotor
for
10
minutes and then resuspended in
%,,th
the volume
of
TED
(50
mmollL Tris HCI, pH
8.0;
I
mmol/L EDTA; and
I
mmotl
L dithiothreitol
[D=]).
The cells were recentrifuged and resus-
pended in the same volume of TED with 0.1 mg/mL of lysozyme
added for
30
minutes. The lysate was sonicated at 4°C three times
with a Branson Soniiier (Branson Ultrasonics, Danbury, CT) using
a
microtip at a power of 6 for
1
minute each cycle. The samples
were centrifuged
for
10 minutes in a Sorvall GSA rotor for l0
minutes at
10,000
rpm, and the supernatants were collected and
stored.
Recombinant proteins were purified from the supernatants of bac-
terial lysates described above by a two-step procedure. Initially, they
were applied to a heparin agarose column (Sigma Chemical CO,
St
Louis, MO), washed with TE buffer (50 mmoln Tris HCl, pH 8.0.
and
1
mmoa EDTA) containing 0.15 moln NaCl and eluted with
TE buffer containing 0.5 mollL NaCl for all the NAP-2 proteins and
1.5
moUL NaCl for all the PF4 proteins. The eluted proteins were
concentrated using YM3 Amico ultrafiltration filters (W.R. Grace
CO,
Beverly, MA), and the buffer was switched to
0.1
%
trifluoroace-
tic acid. The concentrated samples were further fractionated on re-
verse phase high performance liquid chromatography (HPLC).
All purified proteins were run on
20%
precast sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under re-
duced condition followed by Coomassie blue staining. Further identi-
fication was done by enzyme-linked immunosorbent assay (ELBA1
as previously described.” Recombinant protein concentrations were
determined using a Coomassie Protein Assay Reagent Kit with bo-
vine serum albumin as a standard (Pierce, Rockford, IL).
Recombinant 72 amino acid IL-8 protein was purchased (R
&
D
Inc, Minneapolis, MN), and MIP-
la
and
0
were generous gifts from
Drs Barbara Sherry and Anthony Cerami (Rockefeller University,
New York, NY).” Medium containing the CC chemokine C-l0 and
control medium were generously provided by Dr Mark Berger
(Uni-
versity
of
Pennsylvania, Philadelphia).
For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom
CHEMOKINE REGULATION
OF
HUMAN MEGAKARYOCYTOPOIESIS
2561
IL-8Ra and
IL-8RP
Platelet Northern Blot
The IL-8Ra and IL-8RP cDNAs were kindly provided by Dr
Ingrid
U.
SchraufsWer (Scripps Institute, La Jolla, CA) in the ex-
pression vector ~sFFV.neo.~~ The
-
1.2-kb coding regions for both
the IL-8Ra and IL-8RP cDNAs, the 3.3-kb platelet glycoprotein
IIb
(GPIlb), and the 0.4-kb PG cDNA inserts”-38 were released from
their respective vectors by
EcoRI
digestion.
All
of the above cDNA
inserts were purified from an agarose gel using GENECLEAN
(BiolOl, Vista, CA). These inserts were then labeled using (Y-~~P-
dCTP, random primers, and Klenow to
-lo*
CPM/pg DNA.
Total platelet RNA was prepared
from
platelet rich plasma (PRP)
obtained from
100
mL
of sodium citrate anticoagulated blood. Only
the top two-thirds
of
the PRP was used. After spinning down the
platelets, platelet RNA was prepared using the guanidinium thiocya-
natdacid phenol single-step
RNA
isolation te~hnique.’~ The amount
of RNA applied per lane was equivalent to -25% of the total yield.
The RNA was fractionated on gels containing formaldehyde fol-
lowed by transfer to Genescreen Plus membrane (DuPont CO,
Wil-
mington, DE) and hybridization to the labeled probe as previously
described.40
Reverse Transcriptase-Polymerase Chain Reaction
of
Purijied Megakaryocyte
mRNA
Human megakaryocytes were isolated
from
the marrow of remu-
nerated, normal addt volunteers. The isolation
of
greater than
99%
pure megakaryocytes was accomplished as previously described
us-
ing counterflow centrifugal elutriation (Beckman J2-21M; Standard
elutriation motor; Beckman Instruments; Mountainview, CA) to ob-
tain an initial megakaryocyte enrichment:’ followed by isolation
of
morphologically recognizable mature megakaryocytes with a micro-
manipulator. RNA was isolated from
-50
megakaryocytes in less
than 3 hours’ time.
mRNA was isolated from these cells using the Quick-Pre mRNA
Purification Kit (Pharmacia, Piscataway, NJ). The final mRNA pellet
was washed with 75% ethanol and resuspended in
10
pL of triple
distilled autoclaved water. Reverse transcription
of
the megakaryo-
cyte mRNA was performed using
4
pL of the original sample heated
to 65°C for
10
minutes and then cooled on ice for 3 minutes. A total
of 100
U
of Moloney murine leukemia reverse transcriptase
(RT)
(GIBCO BRL, Gaithersburg, MD), 50 ng of random primers (Boeh-
ringer Mannheim, Indianapolis, IN), 40
U
of RNAzin (Promega,
Madison,
WI),
and dNTPs (50 pmoVL each) were added to the tube
and incubated for
1
hour at 37°C. Specific
oligo
primer pairs
used
for
the polymerase chain reaction
(PCR)
amplification with the mega-
karyocyte random cDNA were as follows: II-8Ra: 5“ATGTCA-
AATATTACAGATCC-3’ (sense oligonucleotide;
1
to
20
base,
be-
ginning at the transcriptional start site) and 5”AGATTCATAGAC-
AGTCCCCA-3’ (antisense,
500
to 481 base); and IL-8Rp: 5’-
GAGGACCCAGGTGATCCAGG-3’ (sense, 816 to 835 base) and
5‘-GAGAGTAGTGGAAGTGTGCC-3‘
(antisense, 1065 to 1046
The anticipated PCR products are, therefore, 500 bp and
249 bp for IL-8Ra and
P,
respectively. Ten microliters of the reverse
transcription reaction
or
100
ng of the appropriate IL-8R cDNA or
water was used in a 100-pL PCR reaction using
30
ng of both sense
and antisense primers for each receptor and 2.5
U
Taq polymerase
(Promega) using manufacturer’s supplied buffer and 2.5 pmoVL
MgClz. PCR conditions were melting at 95°C
for
1 minute, annealing
at 60°C, and extension at 72°C for 30 rounds. A total of
10
pL
of
each final PCR product was size-fractionated on a 1.2% agarose gel.
RESULTS
Hematopoietic Effects
of
Recombinant PF4
We first determined whether rH PF4 made in a prokaryotic
system would manifest effects on megakaryocytopoiesis
similar to those we previously reported with highly purified,
serum-derived material. Mature
rH
PF4 protein was synthe-
sized in
Escherichia coli
as described in Materials and Meth-
ods. Its amino acid sequence was identical to the native
protein, except that the recombinant material retained
an
initiating methionine residue.34 Biologic integrity of the engi-
neered protein was demonstrated in chemotactic studies
where the rH PF4 was as effective as serum-derived PF4.34
In a plasma clot assay, both recombinant and native material
inhibited megakaryocyte colony formation in an identical
manner throughout the dose range tested (Fig
1A).
In
agreement with our previously reported results, inhibition
was dose-dependent and most significant at concentrations
2
10
pg/mL.I6 Also
in
agreement with our previous results,
the numbers of cells/colony (Fig 1B) and the size of cells
comprising the colonies (Fig 1C) were also diminished in the
presence of exogenous PF4. Finally, inhibition was mega-
karyocyte lineage specific (data not shown).
NAP-2, AELWPF4 and Mutation Proteins
We have previously shown that PTG does not inhibit in
vitro megakaryocytopoiesis.’6 However, it is now known that
a neutrophil cathepsin G N-terminal cleavage product
of
PTG, a 70-amino acid protein termed NAP-2,
is
a biologi-
cally active form of this protein. NAP-2 is a potent activator
of neutrophils, binding to the IL-8RP on neutrophils.26 To
extend our structure/function studies and to begin to under-
stand the mechanism by which chemokines inhibit megakary-
ocyte development, we tested whether NAP-2, as opposed
to PTG, would inhibit megakaryocytopoiesis. As shown in
Fig 2A, megakaryocyte colony formation was much more
sensitive to the inhibitory effects of NAP-2 than PF4. Where
PF4
inhibited in the
pg/rnL
range, half maximal inhibitory
concentrations of NAP-2 were in the
10
to
100
ng/mL range.
These concentrations were comparable to those needed for
NAP-2 to activate
neutrophil^.^^
As was the case with PF4,
NAP-2’s inhibitory effect was lineage-specific. It has pre-
viously been reported that at PF4 concentrations
2
10 pg/mL
inhibition of CFU-E and Cm-GM might
be
~bserved.”.’~
However, we did not observe such effects
in
our cultures
and
no
definitive changes in CFU-E or CFU-GM colony
formation were observed with increasing doses of NAP-2.
We also sought to determine if NAP-2 inhibited mega-
karyocyte development directly,
or
indirectly via a secondary
effect on accessory marrow cells.
To
address this question,
marrow mononuclear cells were depleted of monocyte-mac-
rophages and T-lymphocytes and then enriched for
CD34+
cells using immunomagnetic beads before exposure to NAP-
2.
Purity of the CD34+ cells in this population consistently
exceeded
85%.
At
50
pg/mL, PF4 decreased the number of
megakaryocyte colonies
to
8%
of the control, and at
750
ngl
mL, NAP-2 decreased expression to
10%
of the control.
These results support the hypothesis that the chemokines
tested exerted a direct inhibitory effect on megakaryocyte
progenitor cells.
Because NAP-2 is thought to activate neutrophils through
IL-8R0, we tested other NAP-2 and PF4 mutant constructs
whose interactions with the neutrophil IL-8 receptors have
been previously defined.33
For
example, alanine substitutions
For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom
2562
GEWIRTZ
ET
AL
I I
I I
I
I
I
I
0
5 10 15 20 25 30 35
40
PF4
@g/ml)
no
PF4
supplement
0
5
10
15 20 25 30 35
40
PF4
(pg/ml)
PF4
supplement
Fig
1.
Inhibition of megakaryocytopoiesis by rPF4.
(A)
Percent decrease
in
the number of colonies seen per
lo5
marrow cells plated at
increasing concentrations
of
PF4.
(01,
rH PF4;
(0).
native PF4.
(B)
The total numbers
of
cells seen per colony
with
increasing concentrations
of rPF4 are shown.
(C)
Immunofluorescence of typical megakaryocyte colonies with (bottom) and without rH PF4 (top), demonstrating the
decrease
in
the size
of
the colony and
of
the megakaryocytes within the colony. Data
in
(A)
and
(B)
represent analysis of
two
or more separate
experiments with four separate plates for each data point. The number of megakaryocyte colonies seen without added chemokine ranged
from
56
to
107
per plate.
at the N-terminus of NAP-2 markedly inhibit its ability
to
activate neutrophils. Interestingly, NAP-2""*, in which the
second amino acid
is
mutated from glutamic acid to alanine
(Table
l),
also
loses its ability to inhibit megakaryocyto-
poiesis (Fig 2B). In contrast, AELRPF4,
in
which PF4's
first eight amino acids proximal
to
the first cysteine residue
are replaced with the four NAP-2 N-amino acid residues
preceding its first cysteine residue, is equivalent
to
NAP-2
in terms of its ability
to
activate neutrophils and to inhibit
megakaryocyte colony formation (Fig 2B).
Inhibitory Effects
of
Other Chemokines
We tested additional chemokines to see whether they too
could inhibit megakaryocytopoiesis.
IL-8
was almost as ef-
fective
as
NAP-2
in
inhibiting megakaryocytopoiesis
in
the
5
to
500
ng/mL concentration range (Fig 3A). Surprisingly,
the
CC
chemokine MIP-la was equally effective (Fig 3A).
Inhibition with both of these chemokines was again noted
to be lineage specific (data not shown). Two additional
CC
chemokines were tested and
also
inhibited megakaryocyto-
poiesis. MIP-
10
decreased megakaryocyte colony formation
to
52%
of the untreated control value at
500
ng/mL, and
a
culture supernatant containing
C-IO,
another member of the
CC
chemokine family, decreased megakaryocyte colonies to
35% of control values at
a
concentration of 2% (vol/vol).
We then sought to determine whether the inhibitory effects
of the
CXC
and
CC
chemokines might be additive
or
syner-
gistic. Progenitor cells were therefore cultured with the indi-
vidual chemokines
at
varying doses,
or
with the two added
together. As shown in Fig 3B, the combination of NAP-2 and
MIP-la had greater inhibitory effect than either chemokine
individually. There does not appear
to
be
a
synergistic effect.
For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom
CHEMOKINE REGULATION
OF
HUMAN MEGAKARYOCYTOPOIESIS
2563
"-1
B
'"7
0'
d
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1'01
1'02
1'0s
1'04
1'0s
1'06
1'07
NAP-2
(ng/ml)
Fig 2. inhibition
of
m.gakaryocytopoi.rh by NAP-2 and
related
constructs. (A) Percant
decr.rw
in
the
number
of
megakaryocyte
colonies aeon par
IC
marrow caih plated at incroasing concentra-
tions of NAP-2.
(01,
CFU-Meg colonies;
(m),
CFU-GM colonies; and
(AI,
CFU-E colonin. PM studlr
at
25 pglmL were
also
done.
(0).
CFU-Mag colonies;
(U),
CFU-GM colonies; and
(A),
CFU-E colonies.
Error bar
nfm
to
OM
standard devlatlon for rmpln studied four
or
mora
times.
IBI
Analyois of CFU-Meg colony numh for PF4
(01.
NAP-2
(01,
NAP-2- (X),
and
AEWPF4 (t).
Data
In (AI and
(B)
analysis of
two
or
mora
.rpernte
exporimento
wlth
four
soparota plates for rch
doto point. The numbor of megakaryocyte
colonin mn without added chemokine ranged
from
51
to
128
per
pinta.
Rather, the combination of
NAP-2
and
MIP-la
better ap-
proximates an additive effect. If the inhibition observed
is
additive, CXC and CC chemokines may inhibit megakaryo-
cyte development by signaling through a common pathway.
It should also
be
noted that even at the highest combined
chemokine dose, megakaryocyte colony formation was not
completely extinguished. When compared with an untreated
control group,
-30%
of maximal colony formation was still
observed even at the highest combined dose level. The nature
of the difference@) between
this
resistant subpopulation and
those progenitor cells that were sensitive to the chemokine
inhibitory effects remains to be determined.
Finally, we also determined whether the chemokine inhib-
itory effect could be abrogated by MGDF stimulation. We
first established that, in our culture system, megakaryocyte
colony growth was maximal at MGDF doses -20 ng/mL.
At such doses, the number of colonies observed in the culture
dishes was comparable to the number observed when optimal
concentrations of aplastic anemia serum plus
L-3
and
E-6
were used as stimulators. We then cultured progenitor cells
in the presence of
50
ng/mL of MGDF and varying doses
of either NAP-2 or MIP-la. As shown in Fig
4,
in compari-
son to the numbers of colonies obtained in the presence of
MGDF alone, NAP-2 at
25
ng/mL and MIF"1a at
>50
ng/
mL
significantly inhibited colony formation. Therefore, even
in the presence of a significant physiologic stimulator, used
at maximally effective concentrations, these chemokines ap-
peared capable of blunting megakaryocyte colony growth.
Demonstration
of
the Expression
of
IL-8Ra
and
IL-8Rp
by
Megakaryocytes
The above studies suggested that chemokines exert a di-
rect effect on megakaryocyte development. If
our
interpreta-
tion of the experiments was correct, it follows that megakary-
ocytes should express the receptors that bind these ligands.
To
provide these important data, we pursued two indepen-
dent, but complementary approaches. First, we isolated total
platelet RNA and looked for
E-8Ra
and
IL-8R@
-A.
The presence of these messages in the total RNA pool would
provide evidence that these receptors might, in fact, be ex-
pressed by megakaryocytes. We also performed RT-PCR for
these same messages on an essentially pure population
of
normal human megakaryocytes.
Platelet
RNA
was extracted
from
the top two-thirds
of
platelet-rich plasma of low-speed centrifuged blood. White
cell contamination was approximately
1
cell per
5,000
plate-
lets. Duplicate lanes of total RNA were hybridized to cDNAs
for the
L-8Ra
and
IL-8RP
and platelet GPIIb and PG as
platelet-specific positive controls. As seen in Fig 5A, there
were single detectable bands
of
the expected size of
-3
kb
for both of the L-8 receptors and
3.3
kb for GPIIb.38.42 An
intense signal was detected with pTG at
0.8
kb with addi-
tional bands seen at
-
1.2 and 1.8 kb.
Total
peripheral blood
neutrophils detect
two
major L-8R bands of
2.4
and
3.0
kb,
Table
1.
Recombinant
PF4
and NAP-2 Chemoklnes
Name Amino Acid Sequence
NAP-2 (wild
type)
AELRCMC
.
.
.
THCN
.
.
.
DGRKICLDPDAPRIKKIVQKLAGDESAD
PF4 (wild
type)
EAEEDGDLQCLC
. .
.
PHCP
. .
.
NGRKICLDLQAPLYKKIIKKLLES
AELWPF4
eCLC.
.
.
PHCP
. .
.NGRKICLDLQAPLYKKIIKLLES
NAP-2EW
+RCMC.
. .
THCN.
.
.
DGRKICLDPDAPRIKKIVQKLAGDESAD
Amino acid differences from wild
type
are underlined.
For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom
2564
GEWIRTZ
ET
AL
while HL60 cell lines only express the
3.0
kb band, similar
to the single band we detected
in
platelet RNA.4'
Even though the blots depicted
in
Fig
5
were hybridized
under high stringency conditions,
it
is possible that we were
only detecting a single IL-8R species because of the
-70%
homology
in
the coding regions of the IL-8R cDNAs. There-
fore, to provide additional proof that we were,
in
fact, de-
tecting both IL-8Ra and IL-gRP, we performed RT-PCR
reactions on mRNA isolated from a small number of
100%
2
-
00
00
-
g g
pure normal human megakaryocytes!3 Primers chosen were
om
33
Oa
Oa
0
7
\a
m=
unique for the two IL-8 receptors, as shown by the fact that
cDNAs (data not shown). These RT-PCR studies demon-
cytes (Fig
5B).
m
0-
2;.a2\azYz
\a
gY
the primers do not yield cross-amplify using IL-8 receptor
5
-
"
d
2.5
IL-8
MIP-1
a
NAP-2
strate that both IL-8 receptors are expressed
in
megakaryo-
okTLXO
Chemokine added (ng/ml)
Fig
3.
Inhibitory
effects
of
CXC
and
CC
chemokines on megakary-
ocytopoiesis. (AI The inhibitory effect of 11-8 and MIP-la on mega-
karyocyte formation relative to an untreated control. NAP2 data
done simultaneously are also included. Data represent the average
of
two
experiments with four plates read for each point
in
each exper-
iment. Error bars refer to one SD. At every concentration shown, the
inhibitory effect was significant at P
c
.003.
(B)
Effect of combined
treatment with NAP-2 and MIP-la. Megakaryocyte colony formation
was tested with NAP-2
(W,
MIP-la
(01,
or both
(XI.
Error bar refers
to
1
SD
for samples studied four or more times. Data
in
(A) and
(El
represent analysis of
two
or more separate experiments with four
separate plates for each data point.
100461
T
0.04
0.009
T
0.y02
S
5'0
500
NAP-2
(ng/ml)
10
DISCUSSION
We have previously shown that PF4, a platelet-specific
CXC chemokine, can directly inhibit
in
vitro megakaryocy-
topoiesis. We now show that other CXC chemokines, NAP-
2,
IL-8, and even the more distantly related CC chemokines,
MIP-la and
P,
and C10, have a similar direct inhibitory
effect
on
in
vitro megakaryocyte development as manifested
by the appearance of fewer colonies composed of smaller
numbers of less mature cells. Inhibition is observed when
chemokines are added to progenitor cell cultures at concen-
trations equivalent to those at which they activate neutrophils
and monocytes,-" suggesting that the inhibitory signals they
generate may be of physiologic significance. Studies per-
formed with mutated NAP-2 and PF4 proteins also support
this hypothesis as the ability
to
activate neutrophils and to
inhibit megakaryocytopoiesis closely parallel each other.
Another highly suggestive finding is that megakaryocytes
express both
a
and isoforms of the IL-8 receptor. Never-
theless, although the presence of these receptors provides a
mechanism for the observed inhibitory effects of some of
the CXC chemokines, further studies will be needed to define
the
full
repertoire of megakaryocyte chemokine receptors
and,
in
particular, whether CC chemokine receptors also
exist on these cells.
0
.L8
5
5'0
560
MIP-la
(ng/rnl)
Fig
4.
Interaction of MGDF and chemokines.
In-
hibitory studies with NAP-2 and MIP-la were per-
formed
in
the presence of
50
ng/mL of rH MGDF.
Data represent the average of
two
experiments with
four plates read for each point
in
each experiment.
Error bars refer to one
SD.
The Pvalue for each study
is shown above the error bar.
For personal use only. by guest on July 13, 2011. bloodjournal.hematologylibrary.orgFrom
CHEMOKINE REGULATION
OF
HUMAN MEGAKARYOCYTOPOIESIS
A
W
l-
c?!
7.5
kb
m
4.4
2.4
t
I
B
M1
2
3123
"
I
L-8
Ra
IL-8RR
Fig
5.
Expression of IL-8Ra and
f3
RNA in megakaryocytes. (A)
Northern blot analysis of total platelet RNA probed with the
cDNAs for pTG, GPllb, IL-8Rrr, and IL-8Rf3.
(B)
Agarose gel of the
RT-PCR products of IL-8Ra and
p.
Lane
1,
mRNA from isolated
megakaryocytes, Lane
2,
the cDNA for appropriate receptor, and
Lane
3,
water control. Size markers are indicated at the side of
both (A) and
(B).
2565
Although many stimulatory cytokines have been described,
relatively few inhibitory proteins have been elucidated. The
best-studied examples of these include the interferons
(INFs),
tumor necrosis factor-a
(TNF-a),
transforming growth factor-
p
(TGF-p), and MP-la.""' The physiologic role of these
cytokines
in
the regulation of human hematopoiesis remains
speculative.44 This is,
in
part, because the biologic effects of
these cytokines are context dependent, a situation that has
tended to generate apparently contradictory reports on their
activity. For example, MP-la appears to specifically inhibit
the growth of cycling hematopoietic cells,46 but when admixed
with IL-3
or
GM-CSF appears to promote colony
growth
in
a
synergistic manner:' In addition,
TNF-a
and the
INFs
appear
to be general suppressers of hematopoietic cell growth, while
the activity of TGF-p is similar to MIP-la
in
that it appears
to block proliferation of growth factor stimulated progenitor
cells, but alone may stimulate CFU-GM and
CFU-E.
These
observations have prompted speculation that some of these
inhibitors,
in
particular TGF-p and MP-la, may play a role
in
maintaining stem cells
in
a
Go
state when hematopoiesis is
otherwise stimulated." In support of this hypothesis,
it
has
been
reported that inhibition of TGF-p expression with antisense
DNA
increases the apparent number of assayable multilineage
progenitor cells
in
cord blood?R These apparently discordant
results may also be explained by the observation that the inhibi-
tory effects of MP-la and TGF-p depend on the maturation
of the progenitor that has been exposed to this chemo-
Many investigators have reported that MP-la
either has no effect
on,
or
actually stimulates the growth
of
more mature progenitors, while it suppresses the growth of
more primitive
C~IIS~~~~~
The fact that the chemokines we have
investigated have no apparent effect on CFU-GM derived col-
ony formation is
in
accord with these
results.
Why the appar-
ently mature CFU-Meg are inhibited by these chemokines when
CFU-GM are not, remains unknown.
Whether the findings we report are of physiologic signifi-
cance remains unclear. The fact that members of this family
can inhibit megakaryocyte development at concentrations
three orders
of
magnitude lower than PF4 is suggestive
of
physiologic relevance because concentrations in this range
are likely achievable
in
vivo. Further, the ability to have
significant inhibition despite maximal levels of
TPO
also
supports a potential in vivo role. Nevertheless, establishing
whether there is a correlation between megakaryocyte/plate-
let mass and chemokine concentrations, either in plasma
or
in
the marrow, will have to be investigated. This will be
particularly important
in
patients with inflammatory states
who may be expected to have relatively high circulating
chemokine levels, and perhaps paradoxically, reactive
thrombocytosis. Measurement of MGDF levels will
be
of
equal importance then, as we have already speculated that
stimulatory growth factors may overdrive the effects of ap-
parently weaker negative regulators.'" Indeed, we have also
hypothesized that negative regulatory loops, whether auto-
crine
or
paracrine
in
nature, are more likely to play a role
in
regulating basal,
or
nonstimulated, megakaryocyte pro-
duction." If shown to be of physiologic significance, these
studies may represent a significant advance
in
the develop-
ment of new pharmacologic strategies to regulate disordered
or
inappropriate thrombopoiesis.
kine.4".49.%1
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2566
GEWIRTZ
ET
AL
ACKNOWLEDGMENT
We thank Dr Pamela Hunt at Amgen Corp (Thousand Oaks, CA)
for the MGDF, Ingrid
U.
Schraufstlitter at the Scripps Institute (La
Jolla, CA) for the E-8R cDNAs, and Dr Mark Berger at the Univer-
sity of Pennsylvania (Philadelphia, PA) for the C10-containing me-
dium.
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