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Increased Serum Levels of Granulocyte Colony-Stimulating Factor in Patients
With Severe Congenital Neutropenia
By Kerstin Mempel, Torsten Pietsch, Thomas Menzel, Cornelia Zeidler, and Karl Welte
Severe congenital neutropenia (SCN), also known as Kostmann Syndrome, is characterized by a maturation arrest of
myelopoiesis at the level of promyelocytes with absence of
neutrophils in bone marrow (BM) and blood. Hypotheses of
the pathophysiology of SCN include (1) defective production
of granulocyte colony-stimulating factor (G-CSF), and/or (2)
defective response to G-CSF. To exclude defective G-CSF
production we tested sera from patients with SCN for the
presence of G-CSF using Western blot analysis and NFS-60
proliferation assay. Using these assays we were able to
detect increased G-CSF serum levels in SCN patients (150 to
910 pg/mL) as compared with normal controls (between
undetectable and 100 pg/mL). These results suggest that
patients with SCN have no defect in G-CSF production but a
defective response of neutrophil precursors to endogenous
G-CSF.
o 1991 by TheAmerican Society of Hematology.
S
neutralizing monoclonal anti-G-CSF antibody 75A was kindly
provided by Dr Souza (Amgen, Thousand Oaks, CA).
Westem blot analysis. Serum G-CSF was partially purified and
concentrated by loading 1 mL aliquots of serum on Sep-Pak C18
columns (Waters, Eschborn, Germany) equilibrated with buffer A
(0.1 mol/L ammonium acetate, pH 4.0). Columns were washed
sequentially with 2 mL buffer A, then with 1 mL buffer A
containing 25% 2-propanol, and subsequently with 1 mL of buffer
A containing 50% propanol. The last 0.75 mL eluting from the
column were collected and lyophilized. For Western blot analysis
the lyophilized sample was dissolved in 10 p L sample buffer (0.06
mol& Tris-HC1 pH 6.8, 2% sodium dodecyl sulfate [SDS], 10%
glycerol, 5% 2-mercaptoethanol). Four microliters were loaded on
a precasted SDS-polyacrylamide gel (Phast System; Pharmacia,
Freiburg, Germany). After electrophoresis, the proteins were
blotted onto nitrocellulose filters by capillary suction for 2 hours at
70°C and then transferred into blocking buffer (0.05 moVL TrisHCI pH 7.4, 5% human serum, 0.5% NP-40, 0.1% Triton-X
[Sigma, St Louis, MO], 0.02% NaN,) overnight at 4°C. For
immunodetection, the filters were incubated with a 1:1,000dilution
of TM-8260 ascites for 45 minutes at room temperature, followed
by a sequential incubation with goat antimouse IgG and alkaline
phosphatase/anti-alkaline phosphatase (APAAP)complex’ (Dianova, Hamburg, Germany) for 45 minutes each. The incubation
with the secondary antibody and APAAP complex was repeated
twice for 10 minutes each. Between incubations, the filters were
washed with blocking buffer. The filters were then developed with
nitroblue-tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) (both from Sigma). Various concentrations of
rhG-CSF8 (Amgen) were diluted in RPMI 1640 (GIBCO, Paisley,
Scotland) + 10% fetal calf serum (FCS) and processed as described above for serum and used as controls. The amount of serum
G-CSF was calculated by comparing the intensity of the immunoreactive and stained protein bands (subsequently called “immunostained” bands) with the immunostained bands of standard dilutions (see above) of rhG-CSF by a video-densitometer (Biotec
Fischer, Reisskirchen, Germany). Using this method the detection
limit of serum G-CSF is 5 pg/mL.
EVERE CONGENITAL neutropenia (SCN; Kostmann
Syndrome), first described by R. Kostmann in 1956,’ is
a disorder of myelopoiesis characterized by an impairment
of myeloid differentiation in BM, absolute neutrophil
counts (ANC) consistently below 200/mm3 in peripheral
blood and onset of severe bacterial infections during the
first 12 months of life.’” The etiology of SCN is unknown.
Clinical phase 1/11 trials with recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been initiated:.’ In our clinic, 30 patients (ages from 2 months to 21
years) from all over Europe have been treated with rhGCSF (3 to 120 bgikg/d) for 3 to 28 months. rhG-CSF was
capable of inducing and maintaining an ANC of above
l,OOO/pL in 29 of 30 patients. This result suggests a defect in
the endogenous G-CSF production or a defective G-CSF
response in these patients. In the present study we show
that patients with SCN are capable of synthesizing and
secreting biologically active G-CSF.
MATERIALS AND METHODS
Patients. Serum samples from 10 patients with SCN, and 19
serum samples from seven healthy volunteers (aged between 18
and 35 years, two men and five women), were obtained before and
during treatment with rhG-CSF. At the time sera were obtained for
G-CSF measurements, no severe bacterial infection was present.
To determine the individual variability of the G-CSF serum levels,
one healthy control was tested weekly for 12 weeks. Three
hematologically normal children with bacterial infections (septicemia, bronchitis, meningitis) were also tested for comparison. The
characteristics of the SCN patients are listed in Table 1. All
patients fullfilled the criteria of SCN: (1) ANC below 200/pL; (2)
maturation arrest of myelopoiesis at the promyelocyte level; (3)
absence of anti-neutrophil antibodies; (4) history of frequent
episodes of severe bacterial infections; and (5) diagnoses in the first
year of life. Serum was separated by centrifugation shortly after
collection and all samples were stored frozen at -80°C until
analysis.
Monoclonal antibodies (MoAbs). The murine MoAb TM-8260
was produced by a protocol as described! After immunization of
(Balb/c X C57BI/6) F, mice, the spleen cells were fused with
murine myeloma cells (P3-NSI-Ag-1) by polyethylene-glycol (PEG
4000; Merck, Darmstadt, Germany). The hybridoma clone TM8260 was detected in an anti-G-CSF enzyme-linked immunosorbent assay. This clone, which produces anti-G-CSF-specific IgG,
antibodies, was further subcloned. TM-8260 ascites was produced
by injecting approximately 5 X lo6 hybridoma cells into the
peritoneal cavity of pristane-primed Balblc mice. Ascites was
cleared by centrifugation and stored in aliquots at -80°C. The
Blood, Vol77, No 9 (May 1). 1991: pp 1919-1922
From the Department of Pediatric Hematology and Oncology,
ChildrenS Clinic, Hannover Medical School, Hannover, Germany.
Submitted November 14,1990; accepted January 8,1991.
Address reprint requests to Karl Welte, MD, PhD, Kinderklinik der
Medizinischen Hochschule Hannover, Konstanty-Gutschow-Str. 8,
0-3000 Hannover 61, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971/91/7709-0019$3.0010
1919
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
MEMPEL ET AL
1920
Table 1. Patient Characteristics
ANClmm'
Before
Patient
Initials
Age
(yl
Sex
Pat i c n t
A MW
Response to
rhG-CSF
rhG-CSF
Therapy'
Therapy?
rh
(X10-3)
C-CSF
BP
FR
NB
1105
106
109
TS
111
KGl
112
KGo
117
HC
118
105
#06
807
~
04
05
06
09
11
12
16
17
18
28
TL
BP
FR
NE
TS
KGI
TG
KGo
MC
MV
9
9
20
6
5
21
17
18
1
8
M
F
M
F
F
M
M
M
F
F
40
32
20
0
0
164
45
420
0
0
Yes (10)
Yes (3)
Yes (3)
NOS
Yes (120)
Yes (3)
Yes (3)
Yes (3)§
Yes (50)
Yes (10)
The SCN was diagnosed in all patients in the first 9 months of life.
'ANC values listed were observed at the times when G-CSF measurements were performed.
tThe numbers in brackets show the doses of rhG-CSF (pglkgldl
required to achieve a complete response (ANC > 1,OOOlpL).
*Patient 9 did not respond to rhG-CSF up to a dose of 60 Fglkgld
subcutaneously or 120 Fglkgld continuous intravenously.
§Patient 17 had at the day before rhG-CSF treatment an ANC of
420lpL.However, in a great number of measurements(n > 100)during
his 17 years of life, the majority of ANCs were below 200lpL.
NFS-60 proliferation ussuy. The murine myeloblastic leukemia
cell line, NFS-60' (kindly provided by W. Farrar, National Cancer
Institute, Frederick, MD), was used to determine G-CSF levels in
sera from patients with SCN. Serial dilutions of sera and appropriate controls (rhG-CSF) were incubated with NFS-60 cells (IVlmL)
for48 hours in 96-well flatbottom microtiter plates (Nunc, Roskilde,
Denmark; 200 pL/well). Identical samples were also tested in the
presence of the neutralizing anti-G-CSF antibody 7SA (4 pg/mL).
'H-thymidine (0.5 pCi/well; Amersham-Buchler, Braunschweig,
Germany) was added for the last 4 hours of culture. Cells were then
lysed and DNA harvested on glass fiber strips. Incorporated
radioactivity was measured in a liquid scintillation counter. Serial
dilutions of rhG-CSF were used as standards, the concentrations of
the samples were calculated from the standard curve by probit
analysis and shown in picograms per milliliter.
14.3-
Control
B Mw
rh
~
(~10-3)
~~
G-CSP
102
#01
#03
#04
Fig 1. Western blot analysis of sera from SCN patients (A) and
controls (E). rhG-CSF (0.5 pg/mL) was used as positive control (left
lanes). Sera were processed as described in Materials and Methods.
trations of all paticnts tcstcd arc listcd in Tablc 2. Using
this method, the serum G-CSF Concentration ranged bctween 150 and 910 pg/mL (Fig 2; Tablc 2). To tcst the
individual variability of the G-CSF serum lcvcls, onc
healthy control was tested weekly for 12 wccks for the
presence of G-CSF in the serum. In this experiment, the
G-CSF concentration ranged betwccn 10 and 25 pg/mL
(median 14 pglmL).
Sera from six patients (Nos. 4, 6, 9, 12, 16, and 28) were
also invcstigatcd for G-CSF activity using the NFS-60
proliferation assay." Sera from SCN paticnts induced significantly highcr proliferation of NFS-60 cells as compared
with sera from controls (Table 3). From thcsc prolifcration
data the conccntration of G-CSF was calculated to be
RESULTS
Sera from nine SCN paticnts (Nos. 4,5, 6, 9, 11, 12, 16,
17, and 18), three patients with bacterial infections, and 19
serum samples from seven healthy controls were collected,
and loaded onto a Sep-Pak C18 column. Proteins eluted
with 25% to 50% propanol and subsequently lyophilized
were analyzed for the prcsence of G-CSF by Western blot
analysis. This method resulted in a partial purification and
approximately 100-fold concentration of serum G-CSF.
Immunostaindcd bands with an apparent molecular weight
(MW) of 19,600 d could bc dctccted in all patients and
some healthy volunteers (Fig 1A and B). rhG-CSF (MW,
18,800 d) was used as a control. The calculation of thc
G-CSF amount was performcd by comparing the density of
the immunostained bands at MW 19,600 with the immunostained bands of serial dilutions of rhG-CSF (MW 18,600)
using a computerized video-densitometer. The generated
graphs of the scanned immunostained protein bands from
serial dilutions of rhG-CSF and from the serum of one
patient are shown in Fig 2. The calculated G-CSF concen-
Pat. $12
rhC-CSF
10
100
300
1000
350
pg/ml
Fig 2. Densitometry of immunostained bands of Western blot
analyses of rhG-CSF (10. 100, 300, and 1,000 pg/mL in RPMl
1640 + 10% FCS) and serum from patient 12. The densitometric
measurementswere performed using a computerized video-denskometer.
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
Table 2. G-CSF Concentrations of Serum Samples From Patients
With SCN (Western blot analysis)
Patient
Initials
rhQ-CSF
Patient
therapy
#04 TL
prior
G-CSF (pglmL)
150
200
220
890
800
350
300
910
320
u-100
280-310
prior
during
#16 TG
prior
=
1
i
DISCUSSION
Table 3. G-CSF Concentrationof Serum Samples From PatientsWith
SCN (proliferationof NFS-60 cells)
Inhibition by
Anti-G-CSF
Antibody.
Patient
Initials
(pg/mL)
( 0 4
04
06
09
12
16
28
TL
FR
NB
KGI
TG
150
250
670
350
300
160
ND
100
60
ND
ND
100
assay
I
Western blol analysis
..&.,
I
during
300
200
=
100
0-CSF lpglmll
SCN is a disorder of myelopoiesischaracterized by severe
neutropenia secondary to maturation arrest of the neutrophil precursors at the level of pr~myelocytes.~.'
Hypotheses
currently discussed for the pathomechanism of the lack of
neutrophils include the absence of G-CSF in patients with
SCN as well as a defective G-CSF response. Patients with
SCN have been shown to respond to administered rhG-CSF
with a dose-dependent increase in their blood neutrophil
The rhG-CSF dosages needed to achieve an ANC
above 100O/pL spanned a wide range (3 to 120 pg/kg/d),
suggesting the underlying defect may be heterogeneous in
nature. In a previous study we were able to show that
Abbreviation: ND, not determined.
'MoAb 75A (IgG,), 4 pg/mL.
1
400
higher in patients (150 to 670 pg/mL) than in controls
(undetectable to 100 pg/mL). The addition of neutralizing
monoclonal anti-G-CSF antibody to these assays reduced
the biologic activity of the G-CSF containing sera by 60% to
100% (Table 3).
In addition, we also measured the G-CSF content of sera
from three patients at various time points during the
rhG-CSF treatment. The data from these three patients are
shown in detail in Fig 3. The serum G-CSF levels of these
patients were significantly lower than to the values before
rhG-CSF treatment and were within the normal range of
healthy individuals (tested with both methods; Fig 3).
MV
112 KGI
durmg
#12 KGI
#16 TG
Abbreviation: U, undetectable.
*Nineteen serum samples from seven individuals.
tsepticemia, meningitis, bronchitis.
G-CSF
1 0 4 TL
NFS-E0 Drollferatlon
1[ - - - I
04
TL
05
BP
06
FR
09
NB
11
TS
12
KGI
16
TG
17
KGo
18
MC
Controls" (n = 19)
Children with bacterial infections (n = 3)t
-1921
G-CSF SERUM LEVELS IN SCN PATIENTS
0
196-'
MoIecu1e.r welght l k O l
Fig 3. G-CSF serum levels of three patients with SCN before and
during rhG-CSF therapy. Left panel, NFS-60 proliferationassay; right
panel, corresponding Western blots. The ANC values at the times
when G-CSF serum level measurements during rhG-CSFtherapy were
performed were 2,712/pL for patient 4, 4,78O/&L for patient 12, and
1,78O/pLfor patient 16.
lipopolysaccharide-stimulatedmonocytes/macrophages from
SCN patients were capable of producing G-CSF." This
report demonstrates that sera from patients with SCN
contain biologically active G-CSF. This result could be
shown in Western blot analyses using a monoclonal anti-GCSF antibody as well as in G-CSF bioassays using the
NFS-60 cell line. The G-CSF levels measured by both
methods in identical serum samples were comparable,
excluding that other cytokines might have influenced the
NFSdO bioassays. As shown in the Western blot analysis,
patients' G-CSF was of the same apparent MW as natural
G-CSF." The MW as shown is 19,600 d. However, this
method does not exclude point mutation of the G-CSF
protein. The G-CSF serum levels in SCN patients are even
higher when compared with serum levels in healthy controls. Initial studies in children with nonhematologic diseases such as bacterial infections also demonstrated elevated G-CSF levels when compared with our control group
(Table 2). In patients with neutropenia (ANC <2OO/pL)
after autologous or allogeneic BM transplantation (BMT)
the G-CSF serum levels were also elevated to values of 100
to 1,OOO pg/mLI2(unpublished data). The data from these
studies (SCN patients and BMT patients) suggest that the
absence of neutrophils leads to a compensating increase in
serum G-CSF levels. This theory is supported by the
observation in both patient groups that as soon as the ANC
is above l,OOO/pL the serum G-CSF levels decrease to levels
below 100 pg/mL1' (Fig 3; unpublished observation). In
both patient groups, the increased G-CSF serum levels
could be explained by upregulation of G-CSF production
by, eg, endothelial cells or fibroblasts due to a feedback
regulation induced by the lack of neutrophils. Secondly,
bacteria, colonizing in higher numbers patients with chronic
neutropenia than controls, may additionally induce G-CSF
production and therefore increase the serum G-CSF levels.
This theory is supported by data from patients with normal
From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
MEMPEL ET AL
1922
ANC but severe bacterial infections demonstrating increased serum G-CSF levels: (Table 2). Thirdly, the high
G-CSF levels could also result from a decreased G-CSF
binding due to the lack of sufficient numbers of responder
cells.
In patients with SCN the underlying pathomechanism for
the lack of neutrophils is therefore not a defective production of G-CSF but might be rather a defective response of
neutrophil precursors to G-CSF. It is possible that the
G-CSF receptors on neutrophil precursors in SCN patients
may have a reduced binding affinity to G-CSF, the number
of receptors may be reduced, or the intracellular signal
transduction may be defective. This theory is supported by
the finding in ongoing clinical trials with rhG-CSF in SCN
patient^:^ that pharmacologic doses as high as 3 to 120
pgikgld are necessary to lead to an ANC of 1,00O/~Lor
more in the majority of patients. These doses induced an
ANC of more than 20,00O/~Lin both primates and cancer
patient^.'^.'^ We cannot exclude that the defect in SCN
patients may be unrelated to G-CSF response, but may
involve a distinct cooperating factor required for terminal
neutrophil production, or perhaps an extracellular matrix
component that is normally required to concentrate and
present G-CSF to myeloid progenitors in the marrow.
In conclusion, the presented data suggest that patients
with SCN have no defect in G-CSF production.
ACKNOWLEDGMENT
We thank Birgit Teichmann for excellent technical assistance
and Angela Schober for secretarial help.
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4. Welte K, Zeidler C, Reiter A, Muller W, Odenwald E, Souza
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From www.bloodjournal.org by guest on February 11, 2015. For personal use only.
1991 77: 1919-1922
Increased serum levels of granulocyte colony-stimulating factor in
patients with severe congenital neutropenia
K Mempel, T Pietsch, T Menzel, C Zeidler and K Welte
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