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Granulocyte Colony-Stimulating Factor Induces hFcyRI (CD64 Antigen)Positive Neutrophils via an Effect on Myeloid Precursor Cells
By J . Martijn Kerst, Jan G.J. van de Winkel, Andrew H. Evans, Masja de Haas, lneke C.M. Slaper-Cortenbach,
Ton P.M. de Wit, Albert E.G.Kr. von dem Borne, C. Ellen van der Schoot, and Rien H.J. van Oers
In this study we have examined hFcyRl expression during
myelopoiesis. Normal bone marrow (BM) cells were found
to express hFcyRl up to the metamyelocyte stage. A different FcyRl expression pattern was observed in an in vitro
model of myelopoiesis. Purified CD34-positive BM cells,
cultured for 1 2 t o 14 days with granulocyte colony-stimulating factor (G-CSF), differentiate into a population of
mature granulocytic cells. Inthese cultures, in which hFcyRl
was virtually absent on the initial CD34-positive BM cells,
hFcyRl was strongly induced by G-CSF after only 5 days.
During final maturation the cells remained hFcyRl positive.
This expression was confirmed functionally by antibodysensitized erythrocytes (EA)-rosette assays. Moreover, the
mature myeloid cells were found to express mRNA encoding
for hFcyRI, whereas reverse-transcriptase polymerase
chain reaction analysis showed that both hFcyRlA and
hFcyRlB genes were expressed. In contrast, on peripheral
blood (PB) polymorphonuclearneutrophil leukocytes (PMN)
the in vitro effect of G-CSF as to hFcyRl induction was limited. Therefore, w e conclude that, with respect t o hFcyRl
expression on PMN, G-CSF acts on myeloid precursor cells
rather than on mature cells. This conclusion could be
strengthened by in vivo administration of a single dose of
G-CSF to a healthy volunteer. After a 12-hour lag time,
hFcyRl expressing PMNs were detected in the peripheral
blood. This study shows that hFcyRl is an early myeloid
differentiation marker that is lost during normal final maturation. However, committed myeloid progenitor cells can
be strongly induced by G-CSF t o express hFcyRI, ultimately
resulting in mature granulocytic cells expressing the highaffinity receptor for IgG. This expression may have important consequencesfor the functional capacity of these cells.
0 1993 by The American Society of Hematology.
0
using EA-rosette assays, northern blotting, and polymerase
chain reaction (PCR) analysis. For comparison, we analyzed
the morphology of hFcyRI-expressing cells in freshly isolated
normal BM. Finally, the effect of G-CSF on hFcyRI expression on peripheral blood polymorphonuclear neutrophil leukocytes (PMN) was investigated both in vitro as well as on
in vivo administration of a single dose of G-CSF to a healthy
volunteer. Our data support the concept that the G-CSFinduced expression of hFcyRI on PMN is the result of GCSF action on myeloid precursor cells.
N BLOOD CELLS, a heterogeneous group of receptors
have the capacity to bind the Fc region of IgG. These
receptors, collectively called Fcy receptors, provide an important bridge between the humoral and cellular branches
of the immune
Three classes of Fcy receptors can
be distinguished, hFcyRI (CD64), hFcyRII (CD32), and
hFcyRIII (CD16), respectively. Each class of receptors consists of closely related molecules encoded by different genes:
hFcyRIA, B, and C, hFcyRIIA, B, and C, and hFcyRIIIA
and B. The expression of these three classes of surface molecules on peripheral blood leukocytes has been studied extensively. In the peripheral blood, hFcyRII and hFcyRIII are
widely distributed: hFcyRII can be detected on monocytes,
neutrophils, eosinophils, basophils, B cells, and platelets,
whereas hFcyRIII is found on neutrophils and natural killer
cells.’X2 In the peripheral blood, hFcyRI is expressed exclusively on monocytes, whereas neutrophils can be induced to
express this receptor by interferon gamma (IFN-y), both in
vitro3-’ and in vivo.’ Data on hFcyRI expression patterns
during myelopoiesis are limited. Progressive loss of the highaffinity Fcy receptor during myeloid differentiation has been
reported.” By means of cell sorting experiments of bone
marrow (BM) cells, Ball et all‘ showed that significant percentages of the committed myeloid progenitors (colonyforming unit granulocyte-macrophage) express hFcy RI and
hFcyRII (20%and 66%, respectively). Moreover, granulocytic
precursors up to the level of band forms were found to express
hFcyRI. Interestingly, Repp et all2 reported the occurrence
of hFcyRI expressing granulocytes after in vivo application
of granulocyte colony-stimulating factor (G-CSF), a finding
that could not be explained by an in vitro effect of G-CSF
on circulating granulocytes.
In this report, we studied in detail the hFcyRI expression
pattern during myeloid differentiation. Using an in vitro
model,13in which purified CD34+ BM cellsk4are cultured in
the presence of G-CSF, we assessed expression of Fcy receptors by means of immuno-flow cytometry. Furthermore, in
this system we analyzed the regulation of hFcyRI expression,
Blood, Vol81, No 6 (March 15). 1993: pp 1457-1464
MATERIALS AND METHODS
Preparation of BM cells and CD34-enrichedprogenitor cells. BM
cells were aspirated from patients undergoing cardiac surgery (Dr
Eijsman; Dept of Cardiac Surgery, Academic Medical Center, Amsterdam, The Netherlands) and from hematologic patients in complete
remission whose disease did not involve the myeloid lineage (no differences in growth rate and differentiation were observed between
BM from these two patients groups). Informed consent was obtained
according to the rules of our hospital. To enrich for CD3Cpositive
cells, mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque;Pharmacia, Uppsala, Sweden; d = 1.077 g/mL).
CD2-, CD19-, and CD67-positive cells were depleted by immunoFrom the Central Laboratory of the Red Cross Blood Transfusion
Service and Laboratory for Experimental and Clinical Immunology,
University of Amsterdam, Amsterdam; the Departments of Immunology and Pulmonary Diseases, University Hospital Utrecht, Utrecht;
and the Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands.
Submitted August 1I, 1992; accepted November 9, 1992.
Address reprint requests to Rien H.J. van Oers, MD, PhD, % Department of Immunohematology, Central Laboratory of the Red Cross
Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam,
PO Box 9190, 1006 AD Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8106-0013$3.00/0
1457
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1458
rosettes and density centrifugation as described.15The enriched cells
were then labeled with fluorescein isothiocyanate (FITC) conjugated
MoAb 8G12 (CD34) and purified by cell sorting (FACStar, Becton
& Dickinson, San Jose, CA). The final cell populations contained
95% to 99% CD34+ cells as determined by flow cytometry.
Morphologic analysis of hFqI2I-expressing cells in BM. Normal
erythrocytedepleted unfractionated BM samples were separated into
hFcyRI-positive and -negative fractions by cell sorting (FACStar).
The fractions were then cytocentrifuged, stained with Jenner-Giemsa
according to standard techniques, and scored microscopically. To
determine the proportion of hFcyR1-positive and -negative cells at
each differentiation stage, the following calculations were performed
the percentage of each cell type in the positive and negative cell fractions was multiplied by the number sorted into that fraction to obtain
absolute cell numbers. The proportions of the specific cell types present in each of the fractions could then be calculated.
Progenitor assay in suspension culture. Five thousand purified
CD34+ cells were plated per 35-mm Petri dish in 1 mL culture medium consisting of Iscove’s modified Dulbecco’s medium (IMDM)
supplemented with 20% (vol/vol) fetal bovine serum, 2 mercapto
ethanol (0.1 mmol/L), penicillin (100 lU/mL), streptomycin (100
pg/mL), bovine serum albumin (BSA; 0.83%, wt/vol), transferrin
(0.77 mg/mL), and glutathione (0.08 mg/mL). Recombinant human
G-CSF (Amgen, Thousand Oaks, CA; 20 ng/mL) or human recombinant granulocyte macrophage colony-stimulatingfactor (GM-CSF;
Sandoz, Uden, The Netherlands: 20 ng/mL) were added at day 0.
After different culture periods at 37°C and 5% COz, cells were collected for immunophenotypical, cytologic, functional, and biochemical analyses.
Neutrophil cultures. Blood was obtained from healthy volunteers
(500 mL blood anticoagulated with 0.4% [wt/vol] trisodium citrate,
pH 7.4), and PMN were carefully purified from buffy coats as described.16On isolation, cells were suspended in IMDM supplemented
with 10%(vol/vol) fetal bovine serum, penicillin (100 IU/mL), and
streptomycin (100 pg/mL) at a concentration of 2 x Io6 cells/mL
and cultured for 12 to 16 hours (37”C, 5% COz) in suspension in
96-well flat-bottomed plates (Nunclon, Roskilde, Denmark; precoated
with 0.5% human serum albumin [CLB] for 1 hour at 37°C). Recombinant human GM-CSF (20 ng/mL), recombinant human GCSF (20 ng/mL), or IFN-y (Boehringer Ingelheim, Mannheim, Germany; 100 U/mL, specific activity: 2 X lo7 U/mg) were added at the
onset of cultures. On culture, cells were harvested and hFcrR1
expression was analyzed by immuno-flow cytometry.
Immunophenotypical analysis. Fcy receptor expression on
CD34’ BM cells at the onset of culture was evaluated as follows: the
BM mononuclear cells were first incubated with a panel of MoAbs.
Subsequently, cells were stained with phycoerythrin-labeled rat-antimouse antibody (Becton Dickinson). The residual binding sites of
the latter antibody were blocked with a mixture of irrelevant murine
antibodies of mlgG1 and mIgG2a isotypes. The CD34 antigen was
detected by FITC-labeled 8G I2 MoAb.
Antigen expression of progenitor cells cultured in suspensions was
analyzed by indirect immuno-fluorescence according to standard
techniques, using a panel of murine MoAbs against monomyeloid
antigens. The following antibodies were used: CLB-HLA-class I1
(HLA-DR), CLB-gran/2 (CD15), B 13.9 (CD67), 197’’ and 32”
(CD64), 1V.3,1941H16” and AT10 (CD32);’ CLB Fc-Cran/lzzand
3GSZ3(CD 16), and FITC-conjugated goat-anti-mouse Ig (CLB GAM
F(ab)2 FITC). The 32, IV.3, and 197 MoAbs were from Medarex
(West Lebanon, NH). The 41H 16 MoAb was a gift from Dr T. Zipf
(University of Texas, Houston, TX). The 8G12 MoAb (CD34) was
a gift from Dr P.M. Lansdorp (Terry Fox Laboratory, Vancouver,
Canada). In control experiments, the anti-IFN-y MoAb, MDI,”
was used. All other MoAbs were produced in our own laboratory
KERST ET AL
and were clustered in the international workshops on Leukocyte Differentiation Antigens.
EA-rosetteassays. Three types of indicator erythrocyteswere used
in rosette assays. Rhesus-D-positive human erythrocytes optimally
sensitized with either a human antiserum against rhesus-D (CLB) or
murine (m) IgG2a or mlgGl anti-glycophorin A switch-variant
MOA^?^ Rosette formation was scored microscopically as described.=
Briefly, to 200 pL cells (2 X IO6 in IMDM, 5% fetal calf serum) either
25 pL medium alone or (in blocking experiments) medium with any
ofthe MoAbs 197, ATIO, IV.3, or 3G8 were added. This suspension
was supplemented with 25 pL of sensitized erythrocytes (2% vol/vol)
and the samples were centrifuged (40g X 4 minutes) and incubated
for 1 hour at 4°C. Cells with at least three erythrocytes bound were
scored as EA-rosettes. Unsensitized erythrocytes (incubated with
phosphate-buffered saline [PBS] as a control) were included in each
experiment and did not form rosettes with any of the cells tested. At
least 200 cells were scored per incubation.
Northern blot analysis. Total cellular RNA was isolated from the
erythroid cell line K562 and from the CD34+ BM cells directly on
isolation, following culture with G-CSF for 11 days, using the RNA
zol B method (Cinna Biotecx, Friendswood, TX).27Ten micrograms
of RNA was then fractionated by electrophoresis, in 1% agarose/
formaldehydegels, and transferred to nitrocellulose (BA85; Schleicher
and Schuell, Dassel, Germany). Hybridization with hFcyR cDNA
probes was performed ovemight in 50% formamide, 3X SSC, 0.1%
sodium dodecyl sulfate (SDS), IOX Denhardt’s solution, supplemented with 50 pg/mL denatured salmon sperm DNA at 42°C. Filters
were washed in 0.3X SSC, 0. I% SDS at 42°C for 30 minutes. A 1.4kb Xho I fragment of cDNA ~ 1 3 5 , ~a ’700-bp Pst I fragment of
cDNA P P W ~ ?and
~ a 900-bp HindIlI/EcoRI fragment of cDNA
F ~ y R l 1 I - were
l ~ ~ used to probe hFcyRI, hFcyRlI, and hFcyRIII,
respectively. Probes were labeled with [ ( U - ~ ~ P I ~ using
C T P a random
primer labeling kit (BRL, Bethesda, MD).
Quantitation of hFcyRI transcripts by reverse transcriptase polymerase chain reaction (RT-PCR). Five micrograms of total RNA,
isolated as described above, was reverse transcribed to cDNA using
Avian Moloney virus reverse transcriptase (1 7 U; Pharmacia) for 90
minutes at 42°C. Incubations were in a total volume of 50 pL, containing 10 mmol/L MgCI2, 100 mmol/L Tris-HCI (pH 8.3), 140
mmol/L KCI, 2 mmol/L DTT, 0.2 mmol/L of each dNTP, and 100
ng of NotI-d(T),* primer (Pharmacia). In most cases, RT reactions
were used directly for PCR amplification, or otherwise stored at
-20°C. Three microliters of RT-cDNA was added to a PCR reaction
(100 pL final volume), which contained 1X PCR buffer (Promega,
Madison, Wl), 0.25 mmol/L each of four dNTP, I O pmol of each
oligonucleotide primer, and 1 U Taq DNA polymerase (PerkinElmer).
Two primers were used to amplify hFcrRI transcripts, primer 9
(5’-ACACCACAAAGGCAGTGA-3’) corresponding to nucleotides
89-106, and primer IO (5’-CACCCAGAGAACAGTGTT-3’) corresponding to the reverse complement of nucleotides 952-969 of cDNA
~ 1 3 5 .Human
~’
actin RT-cDNA was amplified by using primer “5’
actin” (5’-GGCGACGAGGCCCAGAGCAAGAGAGAG-3’) corresponding to nucleotides 204-228, and primer “3’ actin” (5’-TGCCCAGGAAGGAAGGCTGGAAGAG-3’), corresponding to the reverse complement of nucleotides 822-846 of human p actin CDNA.’~
PCR cycles were performed in an automated DNA thermal cycler
(Bio Phase; Bio Excellence, Colchester, UK) using the following temperature profile; denaturation at 94°C for 2 minutes, primer annealing
at 55°C for 2 minutes, and primer extension at 72°C for 3 minutes.
To quantitate the relative amounts of hFcyRl and actin RT-cDNA
in the different samples, 22.5 pL of the PCR products were removed
after 15, 20, 25, and 30 cycles. Ten microliters of each sample were
then fractionated by agarose gel electrophoresis and visualized by
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FcyRl INDUCTION ON MYELOID PRECURSORS BY G-CSF
1459
Blast
Promyelocyte
Myelocyte
Metamyelocyte
Band
PMN
Lymphocyte
Monocyte
0
20
40
60
80
100
120
Fig 1. Morphologic analysis of hFcyRI-expressing cells in normal
BM. Normal erythrocyte-depletedunfractionatedB M samples were
separated into hFcyRI-positive and -negative fractions by cell sorting
(MoAb 32, CD64). Both fractions were then cytocentrifuged, stained
with Jenner-Giemsa, and characterized microscopically. The proportions of hFcyRI-positive (=) and hFcyRI-negative (0)cells per
differentiation stage of B M from three donors 2 SEM are shown.
ethidium bromide staining (see Fig 5A and B). PCR products were
then blotted to nitrocellulose, and specific hFcyRIa and Ib PCRproducts were detected by hybridization with gene-specific oligonucleotides 37A (5’-GAATATCTGTCACTGTGA-3’) and 39B (5’GAATATCACAATACACTG-3’).’I Oligonucleotides were S-phosphorylated with [y12P]adenosine triphosphate (ATP) and polynucleotide kinase (Pharmacia). Actin PCR-product was detected by I2Plabeled, 700-bp EcoRV fragment of human 0 actin cDNA.” Relative
amounts of hFcyRIa (present in 880-bp PCR band”), hFcyRlb2
(present in 600-bp PCR band”), and actin (in the 642-bp PCR product) were quantitated by measuring their incorporated radioactivity
on blots with a Molecular Dynamics 400A Phosphor-imager System
(Molecular Dynamics, Sunnyvale, CA). In some PCR experiments
we observed a third band ( ~ 7 5 bp)
0 at high cycle numbers (>30)in
the hFcyRI-PCR (eg, see Fig 5A), which did not represent a true
transcript and did not affect quantitation of hFcyRla and Ib2 transcripts (at low cycle numbers). To correctly compare the relative
amounts of hFcyR1 and actin RT-cDNAs in the different samples,
only samples present within the range of exponential amplification
were used for quantitation.”
In vivo administration of G-CSF. A single dose of G-CSF (Neupogen, Amgen, 300 pg subcutaneously)was administered to a healthy
volunteer (informed consent was obtained according to the rules of
our hospital). Subsequently, at different time points blood was collected in EDTA for immediate anticoagulation. For erythrocyte lysis,
cells were incubated for 10 minutes with ice-cold isotonic NH4CI
solution (155 mmol/L NH4CI, 10 mmol/L KHC03, 0.1 mmol/L
EDTA, pH 7.4), centrifuged in the cold, and the residual erythrocytes
were lysed during 3 minutes more. The remaining cells were washed
twice in cold PBS/BSA 0.2% (wt/vol) and analyzed by immuno-flow
cytometry.
Statistical ana/.vsis. Student’s t-tests were performed for paired
differences.
RESULTS
Morphologic anal-vsis of hFc/RI-expressing cells in normal
BM. Freshly isolated normal BM cells were separated into
hFcyRI-positive and -negative cells by cell sorting and the
morphology was determined subsequently. In bone marrow
from three individuals, 24.4% k 2.5% of unfractionated BM
cells were hFcyRl positive. Blast cells were detected exclu-
sively in the hFcyRI-negative cell fraction (Fig I). Cells of
intermediate differentiation stages were found to be equally
distributed over the two cell fractions. On maturation into
bands and polymorphonuclears, hFcy RI expression diminished strongly. Lymphocytes were detected in the hFcyRInegative cell fraction, whereas most monocytes were hFcyRl
positive.
The expression of hFcrRI on in vitro generated mature
granulocytic cells. As reported,” culturing of CD34’ BM
cells for 12 to 14 days in the presence of G-CSF results in a
population in which the majority of cells consists of mature
granulocytic cells. This is evident both from morphology (differential counts: myelocytes 4% f I%, metamyelocytes 1 1 %
k 4%, band forms 47% k 6%, and segmented cells 38% f
4%, mean f SEM; n = 4), and immunophenotype (CD15,
75.4% f 0.6%; CD67,70.7% f 2.0%). Interestingly, as shown
in Fig 2, high percentages of these mature day 14 cells were
found to express hFcyRI (63.9% f 3.8%; corrected mean
fluorescence intensity [MFI] 47.5 f 5.3). Both hFcyRII and
hFcyRlll were expressed on 54.4% f 10.6% and 20.5% k
4.2% of the cells, respectively (MR 52.7 ? 13.4 and 17.9 f
7.9). To study the expression of hFcyRI functionally. we
assessed the ability of the cultured cells to bind mlgG2acoated human erythrocytes (EA-mIgG2a rosettes) and antiRhesus D-sensitized erythrocytes. Previous studies have
shown that the murine isotype mlgG2a interacts strongly
with h F ~ y R 1whereas
,~~
Rhesus D-sensitized erythrocytes are
established indicator cells for the high-affinity IgG Fcy receptor.*‘ It was found that 72% f 5% of the day 14 cells
formed mIgG2a EA-rosettes (Fig 3) and 68% f 5% formed
EA-Rh D rosettes (n = 5). The specificity of mlgG2a for
hFcyRI has been established in T-cell proliferation studies,
EA-rosetting, modulation studies, and antibody-dependent
cellular cytotoxicity.’ Inhibition analyses with CD64 MoAb
197 provided additional proof that hFcyRl mediates rosetting
between cultured BM cells and EA-mIgG2a (inhibition:
97.5% f 0.9%, n = 3). A combination of blocking CD32
0
5
9
Culture period (days)
14
Fig 2. Expression of hFcyRl (m), hFcyRll (=I, and hFcyRlll (R)on
CD34+ cells cultured in suspension with G-CSF. After different periodsof culture with G-CSF, Fcr receptor expression was measured
by flow cytometry (MoAbs: 32 [CD64]; IV.3 [CD32]; CLB-gran 1
[CDl SI). Each column represents the mean percentage of positive
cells f SEM of four individual experiments.
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KERST ET AL
1460
t
3
T
0
112
314
516
7-9
Culture period (days)
culture of CD34+ BM cells unequivocally led to a strong
enhancement of both hFcyRIa and Ib2 transcripts (Fig 5C).
The cells generated in vitro also transcribed hFcyRIl and
hFcyRllI genes (Fig 4C and D), although hFcyRIll mRNA
was expressed relatively weakly. The hFcyRIl expressed on
these cells was found to be a product of the hFcyRIlA gene,
as was demonstrated by a polymorphic reactivity with
mIgG’:I evaluated in rosetting experiments with EA-mIgGI.
It was observed that G-CSF stimulated CD34+ BM cells from
some donors effectively formed rosettes (48% & 5%, n = 5)
with EA-mlgG I (high responders). Cells from the remaining
individuals (low responders) did so only weakly (4% & 2%,
n = 3). EA-mlgG 1 rosettes could be blocked completely with
F(ab’)* fragments of CD32 MoAb ATIO. Staining patterns
with 41H 16 (an MoAb that has a unique specificity for the
HR allelic form of hFcyRIIa’’) and IV.3 (recognizing both
allelic forms of hFcyRlla) MoAbs were always found consistent with the EA-mlgG I rosette experiments.
Kinetics of hFcyRI expression during in vitro myelopoiesis. To study the kinetics of hFcyRl expression on
CD34+ cells during culture with G-CSF, cells were harvested
at different time intervals and analyzed for hFcyRl expression
immunophenotypically and functionally. Immunophenotypically, at day 0 the expression of hFcyRl (CD64) and
hFcyRIl (CD32) on CD34’ BM cells was very low, albeit
significantly higher than the controls (6.8% k 2.2% and 9.5%
& 3.1% positive cells). The hFcyRIII (CD16) was undetectable
on the progenitor cells (Fig 2). However, at the onset of culture, no rosette formation was observed between CD34’ BM
cells and EA-mlgG2a (Fig 3). In accordance. by Northern
blot analysis no mRNA for any of the hFcy receptors was
I
14
Fig 3. EA-rosette formation between mlgG2a-sensitized erythrocytes and CD34+ B M cells cultured with GM-CSF (IB)or G-CSF
(m), alone or in combination (a).Each column represents the mean
percentage EA-rosette-forming cells
SEM (n H 5) after different
periods of culture.
(IV.3, mlgG2b) and CD16 (3G8, mlgGI) MoAbs had only
a marginal effect on EA-mlgG2a rosetting (3.2% & 1.5% inhibition, n = 3). As shown in Fig 4A, mRNA for hFcyRl
was abundantly present in these mature cells. Furthermore,
RT-PCR analysis demonstrated that the cells expressed both
hFcyRla and hFcyRIb genes (Fig 5A). The relative abundance of the two transmembrane forms of hFcyRI, la and
Ib23’ was quantitated by using the ubiquitously expressed
actin (Fig 5B) to serve as an internal control for the amount
of RNA tested (semiquantitative PCR). In all three donors,
A FcpI
B
control
+28 S
+18 S
C FcXRII
(u
$
0
D FC~RIII
- --
-
(u
- -u 2
a ? -
l - v -
Y U k ob6
m
Y
-
7
& c j
I
-~
.
+28 S
c28
s
Fig 4. RNA blot analysis of hFwR transcripts.
Total cellular RNA f”freshly isolated CD34+ B M
cells (indicated as A-0) as well as from CD34+ B M
cells on culture with G-CSF for 11 days (three donors; A - l l,B-1l,and c - l l ) , and from K562 cells
(control) were isolated, electrophoresed, blotted,
and hybridized with 3*P-labeledh F c ~ R(A),
l hFcvRll
(C), or hFcrRlll (D) cDNA. (B) Ethidium bromidestaining pattern. Positions of the 28s and 1 8 s ribosomal RNAs are indicated.
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FCyRl INDUCTION ON MYELOID PRECURSORS BY G-CSF
1461
hFc6RI
A-O
A-ll
8-11
c-It
15 20 25 30 15 20 25 30 15 20 25 30 15 20 25 30
v)
t;;2
3a
A
880++
600+
Fig 5. Expression of hFcyRl transcripts in
CD34+ B M cells on culture with G-CSF, analyzed
by RT-PCR. Whole RNA was isolated from CD34+
BM cells from three donors (designated A, B, and
C) directly on isolation (A-0) or culture with G-CSF
for 11 days (A-1 1, 6-11, C-11). RNA was reverse
transcribed, and hFcyRl transcripts were specifically amplified by PCR (15 to 30 cycles). Ethidium
bromide staining of PCR products separated by
agarose gel electrophoresis is shown (A). Amounts
of RT-cDNAs were calibrated by parallel amplification of actin in each sample (6). PCR-products
were blotted to nitrocellulose. The hFcyRla transcripts (present in 880-bp PCR band) were detected by hybridization with an hFcyRIA-specific
primer, hFcyRlb2 (present in 600-bp PCR band) by
a primer selectively hybridizing with 1B geneproducts, as described in Materials and Methods.
The radioactivityof PCR productswas quantitated
with a Phosphorimager System, and data are presented in (C). The relative amounts of hFcyRl transcripts present in uncultured CD34+ cells (A-0)
was set at 1.
B
c
I
15 20 25 30 15 20 25 30 15 20 25 30 15 20 25 30
8 ’1
Relative Abundance of PCR-products
ap h Fc/Rla
0 h FckRlb2
r
4J
detectable in the initial cell populations (Fig 4). As reported
before,” G-CSF induces a sharp increase in proliferation of
CD34+ cells until day 11, after which proliferation declines.
Immunophenotypically, cells cultured with G-CSF downregulate HLA-DR (day 0, 85.6% ? 4.8%; day 14, 10.3% ?
2.0%), whereas subsequently, mature granulocytic antigens
CD I5 and CD67 are upregulated. At day 5 of culture, significant numbers of the cells express CD15 (39.2% ? 2.6%);
a strong induction of CD67 can be observed from day 9 onward (37.9% ? 6.8%). Figure 2 shows the sequence of the
expression of Fcy receptors during this G-CSF-induced in
vitro differentiation of CD34+ cells. The first FcyR to be
expressed was hFcyRI (at day 5 already 31.3% f 4.9%,
reaching levels of 52.8% f 3.4% at day 9 and 63.9% ? 3.8%
at day 14). The hFcyRll appeared somewhat later: at day 5,
only 8.7% f 4.2% of the cells expressed hFcyRII, reaching
a level of 54.4% f 10.6% at day 15. The hFcyRIll was only
expressed on low numbers of cells (day 5,0.8% ? 0.3%; day
15, 20.5% f 4.2%). Soluble hFcyRIII could not be detected
in the culture supernatant using a very sensitive
radioimmunoa~say~~
(data not shown).
Functional analysis of hFcyRI expression during in vitro
maturation showed a strong increase of EA-mIgG2a rosetteforming cells from day 5 or 6 onward (25.7% ? 4.4%, Fig 3),
reaching levels comparable to the percentage of positive cells
as determined by immunoflow cytometry with CD64 MoAb.
Next, the effects of G-CSF were compared with granulocytemacrophage (GM)-CSF. Although GM-CSF also induced an
initial hFcyRI expression, by day 5 or 6 the expression had
A-0
A-ll
8-I
c-ll
reached a plateau of 20% to 30%. In the presence of a combination of GM-CSF and G-CSF, the cells formed EAmlgG2a rosettes more rapidly, but the rosetting pattern was
similar to that in cultures with G-CSF alone.
To exclude the premise that IFN-y, a known potent inducer
of hFcyRl on both monocytes and PMN,’ mediates the effects
on hFcyRI during culture of CD34+ BM cells, we performed
control CD34+ BM cultures in the presence of neutralizing
antibodies against IFN-y (final concentration 5 pg/mL; neutralizing 900 U/mL IFN-yZ4).No effect on G-CSF-induced
hFcyRI expression was observed (percentage inhibition in
two experiments; 3.5% and 5.4%, respectively). Moreover, in
agreement with Zoumbos et al,37addition of exogenous IFNy to cultures strongly inhibited proliferation of G-CSF-cultured CD34’ BM cells (data not shown).
In vitro effeccr ofG-CSFon hFcyRI expression by peripheral
blood (PB) PMN. Purified, freshly isolated PB PMN were
cultured for 12 to 16 hours in the presence of either GMCSF, G-CSF, or IFN-y (Fig 6). GM-CSF did not affect the
expression of hFcyRI (MFI, 2.3 ? 0.9; 3.5% ? 1.2% positive
cells). Compared with PMN cultured in medium alone, a
moderate albeit significant (P< .05) hFcyRI expression was
observed in response to culture with G-CSF (MFI, 6.7 ? 2.8;
23. I % f 7.8%). IFN-y induced a marked upregulation (Pe
.002) of hFcyRI expression (MFI, 14.2 f 2.5; 30.7% f 4.2%).
The expression of hFcyRI on PB PMN on G-CSF administration in vivo. G-CSF (neupogen; 300 pg subcutaneously)
was administered to a healthy volunteer. Figure 7 shows the
hFcyRI expression level on PMN at different time intervals
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1462
KERST ET AL
Unst imulated
GM-CSF
G-CSF
IFN-gamma
0
5
10
15
20
25
Mean Fluorescence Intensity
Fig 6. The hFcyRl expression by PB neutrophils cultured in the
presence of IFN-y, GM-CSF, or G-CSF. Expression was analyzed
flow cytometrically using MoAb 32. Data are presented as average
MFI corrected for MFI of isotype-matched control MoAb (arbitrary
units) of five experiments f SEM.
after G-CSF administration. Until 12 hours had elapsed, only
minimal hFcyRI expression could be detected on PMN.
However, between 12 and 72 hours hFcyRI expression was
strongly increased. After 24 and 48 hours as much as 77%
of the PMNs were hFcyRI positive. Morphologic analysis
demonstrated that always more than 90%of the circulating
granulocytic cells were polymorphonuclears. After 24 hours
these circulating PMN also expressed mRNA coding for
hFcyRL3*
DISCUSSION
hFcyRIII. It has been shown that hFcyRIII is easily shed
from PMN!'
However, soluble hFcyRIII could not be detected in the culture supernatant using a very sensitive radioimmun~assay.~~
Moreover, hFcy RIII mRNA expression
was relatively weak compared with hFcyRI and hFcyRII.
Furthermore, we assessed the kinetics of Fcy receptor
expression during myelopoiesis in vitro. Immunophenotypically, hFcyRI and hFcyRII expression on the initial CD34+
BM cells was low, whereas hFcyRIII was undetectable. Both
functionally and at the level of mRNA, CD34" BM cells did
not express hFcyRI. Culturing with G-CSF resulted in a sequential pattern of Fcy receptor expression on CD34' BM
cells; first hFcyRI, followed by hFcyRII and hFcyRIII, respectively. The presence of hFcyRI was confirmed functionally by using both mIgG2a and Rhesus D-sensitized erythrocytes. EA-mIgG2a rosette analysis of the BM cells cultured
with GM-CSF showed limited hFcyRI expression, consistent
with minimal final maturation observed with this cytokine
(mainly myelocytes and metamyelocytes"). On culture with
a combination of GM-CSF and G-CSF ultimately resulting
in metamyelocytes and band forms,'3 hFcyRI expression,
although appearing earlier in culture, was essentially the same
as with G-CSF alone.
In conclusion, during normal myelopoiesis hFcyRI
expression is found until the metamyelocytic stage. This is
in agreement with findings by Ball et al." However, during
G-CSF-induced in vitro myeloid differentiation, the highaffinity receptor remains expressed during final maturation.
We hypothesize that G-CSF acts at the level of precursor cells
rather than on mature cells, because we and other
investigator^^,^ observed that in vitro G-CSF has only a marginal effect on hFcyRI expression by PMN from PB. Our
data may also provide an explanation for recent observations
by Repp et all2 who reported hFcyRI expression by neutrophils on in vivo application of G-CSF. To test this hypothesis,
we monitored the effect of G-CSF as to hFcyRI expression
on PMN in vivo. In the study of Repp et a1," PMN of car-
In this study we have examined hFcyRI expression during
myelopoiesis. It was found that ex vivo isolated BM cells
express hFcyRI up to the metamyelocytic stage of differentiation. However, during in vitro myelopoiesis, in which
CD34+ BM cells cultured in the presence of G-CSF differentiate into a population of mature granulocytic cells,I3 high
I
I
percentages of these mature cells were found to express
hFcyRI, both immunophenotypically and functionally.
Moreover, the mature cells expressed hFcyRI mRNA. Recently, three genes have been described encoding ~ F c ~ R I . ~ ' , ~ ~
8
RT-PCR analysis showed that hFcyRI mRNA expressed by
the cultured cells represented transcripts of both the hFcyRIA
c 24
gene (encoding hFcyRIa, a transmembrane protein with three
extracellular Ig-like domains) and the hFcyRIB gene
(hFcyRI2, an alternative splice form that encodes a transmembrane protein with two extracellular Ig-like domains3').
In addition, the mature cells were demonstrated to contain
144
96
hFcyRII and hFcyIII transcripts. The hFcyRII expressed by
0
5
10
15
20
25
the cells was encoded by the hFcyRIIA gene, as was supported
by both the polymorphic binding-pattern of EA-mIgG 1 and
Mean Fluorescence Intensity
the differential staining with 4 1H 16 MoAb, which selectively
Fig 7 . The hFcyRl expression on PMN on in vivo application of
interacts with the hFcyRIIaHRallotype.20The presence of the
G-CSF (300 pg, subcutaneously). The expression was analyzed by
hFcyRIIa HR-LR polymorphism on mature granulocytic
flow cytometry using MoAb 32. MFI corrected for isotype-matched
cells derived from CD34+ BM cells cultured with G-CSF is
control MoAb (arbitrary units) at different times following injection
consistent with studies on PB PMN.20,40Remarkably, only
of G-CSF are presented. After 2 4 and 48 hours as much as 77% of
the PMN were hFcyRl positive.
a low percentage of the final mature cell population expressed
I
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
FcrRl INDUCTION ON MYELOID PRECURSORS BY G-CSF
cinoma patients were evaluated 3 days after the start of continuous G-CSF therapy. We now studied the time course of
FcRI expression after administration of a single dose G-CSF
(300 pg, subcutaneously) to a healthy volunteer. PMN expressing hFcyRI began to appear in the PB no sooner than
12 hours after administration, and maximal hFcyRI expression was observed after 24 hours. This lag-time is consistent
with the in vitro findings that G-CSF does not induce hFcRI
expression on mature granulocytes, which have a peripheral
half-life of only 7 to 8 hours, an interval that has been shown
not to be affected by G-CSF!2 Morphologic analysis of circulating cells in the volunteer tested showed more than 90%
of the PB granulocytic cells to be polymorphonuclear. Because
BM PMN were found to be hFcyRI negative (Fig I), the
release of storage pool granulocytes is unlikely to be responsible for the observed hFcyRI expression on PMN. Thus,
our results support the hypothesis that myeloid precursors
are the target cells and are in agreement with the observation
that G-CSF reduces marrow transit time of the maturating
myeloid population (1 day as compared with 4 to 5 days).42
Interestingly, hFcyRIII expression on PMN was found to be
strongly decreased on in vivo application of G-CSF.43Also,
during in vitro maturation we observed only low percentages
of the mature cells expressinghFcyRIII. Decreased expression
of this PMN surface marker on G-CSF treatment of severe
congenital neutropenia patients has been reported previously.44
In our opinion, hFcyRI represents an early myeloid differentiation antigen that is lost during normal final maturation. On in vivo application of G-CSF, hFcyRI remains
present during the terminal differentiation process toward
mature circulating PMNs. This may also account for the observed presence of hFcyRI on PMN in patients with streptococcal pharyngitis45because increased levels of G-CSF have
been found during bacterial infections!6 The biological significance of the remarkable transient hFcyRI expression
during intermediate differentiation stages remains to be elucidated. Interestingly, transient Fcy receptor (I1 and 111)
expression has been reported during thymocyte differentiation!'
The expression of hFcyRI has been reported to trigger
multiple biological functions.' In vivo administration of GCSF can activate effector functions in mature PMN.48Thus,
the G-CSF-induced abundant expression of high-affinity receptors for IgG on PMN may possibly provide these cells
with an optimal capacity to perform all known functions
mediated by Fc receptors.'~~~
ACKNOWLEDGMENT
We gratefully thank Berend Hooibrink for skillfully operating the
FACStar, Dr Paul W.H.I. Parren for providing anti-glycophorin-A
switch-variants, Marion Kleyer for measurement of soluble CD 16,
and Tessy Emst for cooperation in some of the experiments.
REFERENCES
1. Van de Winkel JGJ, Anderson CL: Biology of human immunoglobulin G Fc receptors. J Leukoc Biol49:51 I, 1991
2. Ravetch JV, Kinet J-P: Fc receptors. Ann Rev Immunol9:457,
1991
1463
3. Perussia B, Dayton ET, Lazarus R, Fanning V, Trinchieri G:
Immune interferon induces the receptor for monomeric IgGl on
human monocytic and myeloid cells. J Exp Med 158:1092, 1983
4. Petroni KC, Shen L, Guyre PM: Modulation of human polymorphonuclear leukocyte IgG Fc receptors and Fc receptor-mediated
functions by IFN-gamma and glucocorticoids. J Immunol 1403467,
1988
5. Buckle AM, H o g N: The effect of IFN-gamma and colonystimulating factors on the expression of neutrophil cell membrane
receptors. J Immunol 143:2295, 1989
6. Buckle AM, Jayaram Y, H o g N: Colony-stimulating factors
and interferon-gamma differentially affect cell surface molecules
shared by monocytes and neutrophils. Clin Exp Immunol 81:339,
1990
7. Erbe DV, Collins JE, Shen L, Graziano RF, Fanger M W The
effect of cytokines on the expression and function of Fc receptors for
IgG on human myeloid cells. Mol Immunol 2757, 1990
8. Pan L, Mendel DB, Zurlo J, Guyre PM: Regulation of the steady
state level of Fc-gamma-mRNA by interferon gamma and dexamethasone in human monocytes, neutrophils and U-937 cells. J Immunol 145:267, 1990
9. Huizinga TWJ, van der Schoot CE, Roos D, Weening RS: Induction of neutrophil FC-gamma receptor I expression can be used
as a marker for biologic activity of recombinant interferon-gamma
in vivo. Blood 77:2088, 1991
10. Fleit HB, Wright SD, Dune CJ, Valinski JE, Unkeless JC:
Ontogeny of Fc receptors and complement during human myeloid
differentiation. J Clin Invest 73516, 1984
1 1 . Ball ED, McDermott J, Griffin JD, Davey FR, Davis R,
Bloomfield CD: Expression of three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies
on normal bone marrow and acute leukemia cells. Blood 73:1951,
1989
12. Repp R, Valerius Th, Sendler A, Gramatzki M, Iro H, Kalden
R, Platzer E: Neutrophils express the high affinity receptor for IgG
(FcRI, CD64) after in vivo application of recombinant human granulocyte colony-stimulating factor. Blood 78:885, 199 1
13. Kerst JM, Slaper-Cortenbach ICM, von dem Borne AEGKr,
van der Schoot CE, Van Oers MHJ: Combined measurement of
growth and differentiation in suspension cultures of purified human
CD34 positive cells enables a detailed analysis of myelopoiesis. Exp
Hematol 20: 1 188, 1992
14. Civin CI, Banquerigo ML, Strauss LC, Loken MR: Antigenic
analysis of hematopoiesis. VI. Flow cytometric characterization of
My- IO-positive progenitor cells in normal human bone marrow. Exp
Hematol 15:10, 1987
15. Slaper Cortenbach IC, Admiraal LG, van Leeuwen EF, Kerr
JM, von dem Borne AE, Tetteroo PA: Effective purging of bone
marrow by a combination of immunorosette depletion and complement lysis. Exp Hematol 18:49, 1990
16. Verhoeven AJ, van Schaik MJL, Roos D, Weening RS: Detection of carriers of the autosomal form of CGD. Blood 71505,
1988
17. Guyre PM, Graziano RF, Vance BA, Morganelli PM, Fanger
MW: Monoclonal antibodies that bind to distinct epitopes on FcRI
are able to trigger receptor function. J Immunol 143:1650, 1989
18. Anderson CL, Guyre PM, Whitin JC, Ryan DH, Looney RJ,
Fanger M W Monoclonal antibodies to Fc receptors for IgG on human
mononuclear phagocytes: Antibody characterization and induction
of superoxide production in a monocyte cell line. J Biol Chem 26 1:
12856, 1986
19. Looney RG, Abraham GN, Anderson C L Human monocytes
and U937 cells bear two distinct Fc receptors for IgG. J Immunol
136:1641. 1986
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1464
20. Gosselin EJ,Brown MF, Anderson CL, Zipf TE, Guyre PM:
The monoclonal antibody 4 1H 16 detects the Leu-4 responder phenotype of human FcgammaRII. J Immunol 144:1817, 1990
21. Greenman J, Tutt AL, George AJT, Pulford KAF, Stevenson
GT, Glennie MJ: Characterization of a new monoclonal antibody,
ATlO, and its incorporation into a bispecific F(ab’), derivate for recruitment of cytotoxic effectors. Mol Immunol 28: 1243, 1991
22. Wemer G, von dem Borne AEGKr, Bos MJE, Tromp JF,
Van der Plas CM, Engelfriet CP, Tetteroo PAT: Localization of the
human NA I alloantigen on neutrophil Fc-gamma receptors, in
Reinherz EL, Haynes BF, Nadler LM, Bemstein ID (eds): Leukocyte
Typing 11, vol 3. New York, NY, Springer-Verlag, 1986, p 109
23. Fleit HB, Wright SD, Unkeless JC: Human neutrophil Fcgamma receptor distribution and structure. Proc Natl Acad Sci USA
79:3275, 1982
24. Van der Meide PH, Dubbeld M, Schellekens H: Monoclonal
antibodies to human immune interferon and their use in a sensitive
solid phase ELISA. J Immunol Methods 79:293, 1985
25. Boot JAH, Geerts M U , Aarden LA: Functional polymorphisms of Fc receptors in human monocyte-mediated cytotoxicity
towards erythrocytes induced by murine isotype switch variants. J
Immunol 142:1217, I989
26. Van de Winkel JGJ, Tax WJM, van Bruggen MCJ, van Roozendaal CEP, Willems HW, Vlug A, Capel PJA, Koene RAP Characterization of two Fc receptors for mouse immunoglobulins on human monocytes and cell lines. Scand J Immunol26:663, 1987
27. Kindt GC, Van de Winkel JGJ, Moore SA, Anderson C L
Identification and structural characterization of Fc receptors on pulmonary alveolar macrophages. Am J Physiol26O:L 403, I99 l
28. Allen JM, Seed B: Isolation and expression of functional highaffinity Fc receptor complementary DNAs. Science 243:378, 1989
29. Warmerdam PAM, Van de Winkel JGJ, Gosselin EJ, Capel
PJA: Molecular basis for a polymorphism of human Fc receptor I1
(CD32). J Exp Med 172:19, 1990
30. Ravetch JV, Perusia B Altemative membrane forms of FcRIII
(CD16) on human NK cells and neutrophils: Cell-type specific
expression of two genes which differ in single nucleotide substitutions.
J Exp Med 170481, 1989
3 1. Emst LK, Van de Winkel JGJ, Chiu I-M, Anderson C L Three
genes for the human high-affinity Fc receptor for IgG (FcRI) encode
four distinct transcription products. J Biol Chem 267: 15692, 1992
32. Ponte P, Ng SY, Engel J, Gunning P, Kedes L: Evolutionary
conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucleic Acids Res 12:1687,
1984
33. Rupp AW, Weintraub H: Ubiquitous MyoD transcription at
the midblastula transition precedes induction-dependent MyoD
expression in presumptive mesoderm ofX. luevis. Cell 65:927, 1991
34. Jones DH, Looney RJ, Anderson CL: Two distinct classes of
IgG Fc receptors on a human monocytic line (U937) defined by
differences in binding of murine IgG subclassesat low ionic strength.
J Immunol 135:3348, 1985
KERST ET AL
35. Tax WJM, Willems HW, Reekers PPM, Capel PJA, Koene
RAP Polymorphism in mitogenic effect of IgGl monoclonal antibodies against T3 antigens on human T-cells. Nature 304:445, 1983
36. Huizinga TW, de Haas M, Kleijer M, Nuijens JH, Roos D,
von dem Borne AEGKr: Soluble Fc gamma receptor III in human
plasma originates from release by neutrophils. J Clin Invest 86:4 16,
1990
37. Zoumbos NC, Baranski B, Young NS: Different haematopoietic growth factors have different capacity in overcoming the in
vitro interferon gamma-induced suppression of bone marrow progenitor cells. Eur J Haematol44:282, 1990
38. Valerius T, Repp R, de Witt TPM, Berthold S, Platzer E,
Kalden JR, Gramatzki M, van de Winkel JG: Involvement of the
high affinity receptor for IgG (FcyRI; CD64) in enhanced tumor cell
cytotoxicity of neutrophils during G-CSF therapy. (submitted)
39. Van de Winkel JGJ, Emst LK, Anderson CL, Chiu IM: Gene
organization of the high-affinity receptor for IgG, FcgammaRI
(CD64). J Biol Chem 266: 13449, 1991
40. LeeuwenbergJFM, Van de Winkel JGJ, Jeunhomme TMAA,
Buuman WA: Functional polymorphism of IgG FcRII (CD32) on
human neutrophils. Immunology 7 1:301, 1990
4 1. Huizinga TW, van der Schoot CE, Jost C, Klaassen R, Kleijer
M, von dem Bome AEGKr, Roos D, Tetteroo PA: The PI-linked
receptor FcRIII is released on stimulation of neutrophils. Nature
333:667, 1988
42. Lord BI, Bronchud MH, Owens S, Chang J, Howell A, Souza
L, Dexter TM: The kinetics of human granulopoiesis following treatment with granulocyte colony-stimulating factor in vivo. Proc Natl
Acad Sci USA 86:9499, 1989
43. Kerst JM, de Maas M, Kleyer M, van der Schoot CE, Slaper
JCM, von dem Bome AEGKr, van Oers RHJ: In vivo administration
of G-CSF results in both immunophenotypically and functionally
altered neutrophils. (manuscript in preparation)
44. Elsner J, Roesler J, Emmeldorffer A, Zeidler C, LohmannMatthes ML, Welte K: Altered function and surface marker expression
of neutrophils induced by rhG-CSF treatment in severe congenital
neutropenia. Eur J Immunol 48:10, 1992
45. Guyre PM, Campbell AS, Kniffin WD, Fanger MW: Monocytes and polymorphonuclear neutrophils of patients with streptococcal pharyngitis express increased numbers of type I IgG Fc receptors. J Clin Invest 86: 1892, 1990
46. Kawakami M, Tsutsumi H, Kumakawa T, Abe H, Hirai M,
Kurosawa S, Mori M, Fukushima M: Levels of serum granulocyte
colony-stimulating factor in patients with infections. Blood 76: 1962,
1990
47. Rodewald HM, Moingeon P, Lucich JL, Dosiou C, Lopez P,
Reinherz EL: A population of early fetal thymocytes expressingFcRII/
I11 contain precursors of T lymphocytes and natural killer cells. Cell
69:139, 1992
48. Demetri GD, Griffin J D Granulocyte colony-stimulating factor and its receptor. Blood 78:279 I, 1991
49. Lieschke GJ, Burges A W Granulocyte colony-stimulating
factor and granulocyte-macrophage colony-stimulatingfactor. N Engl
J Med 327:28, 1992
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1993 81: 1457-1464
Granulocyte colony-stimulating factor induces hFc gamma RI (CD64
antigen)-positive neutrophils via an effect on myeloid precursor cells
JM Kerst, JG van de Winkel, AH Evans, M de Haas, IC Slaper-Cortenbach, TP de Wit, AE von
dem Borne, CE van der Schoot and RH van Oers
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