1. No category

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1994 84: 789-794
Abnormal response to granulocyte colony-stimulating factor (G-CSF)
in canine cyclic hematopoiesis is not caused by altered G-CSF
receptor expression
BR Avalos, VC Broudy, SK Ceselski, BJ Druker, JD Griffin and WP Hammond
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Abnormal Response to Granulocyte Colony-Stimulating Factor (G-CSF)
in Canine Cyclic Hematopoiesis Is Not Caused by Altered
G-CSF Receptor Expression
By Belinda R. Avalos, Virginia C. Broudy, Sarah K. Ceselski, Brian J. Druker,
James D. Griffin, and William P. Hammond
A decrease in responsiveness t o granulocyte colony-stimulating factor (G-CSF) hasbeen implicated in the pathophysiology of cyclic hematopoiesis. Using the canine model of
cyclic neutropenia,we examined the response of neutrophil
precursors t o G-CSF in vitro and G-CSFreceptor expression
in neutrophils from grey collie dogs t o determine whether
the abnormal response observed to G-CSF in vivo in this
disorder is present at the level of the progenitor cell and is
caused by defective G-CSF receptor expression. Bone marrow mononuclear cells from grey collie dogs required sevenfold higher G-CSF concentrations than normal dog cells
t o achieve half-maximal colony growth (56 pmol/L v 8 pmol/
L).Receptor binding assays with '261-labeledG-CSF and
Scatchard analyses of the equilibrium binding data were
consistent with expression of a singleclass of high-affinity
receptors for G-CSF on neutrophils from both normal dogs
and grey collies with similar receptor numbers (56 to 446
siteslcell W 78 t o 199 sites/cell) and binding affinities (28 to
206 pmol/L v84 to 195 pmol/L). Chemical cross-linkingstud-
ies identified a G-CSF binding protein of approximately 120
kD on neutrophils from grey collies, similar in size t o that
on normal dog neutrophils. No abnormal G-CSF receptor
mRNA transcripts were detected in neutrophils from grey
collie dogs by Northern blot analysis. Treatment of both
normal and grey collie neutrophils with G-CSF rapidly induced tyrosine phosphorylation of an 80-kDprotein that behaved like canine c-rei. These results demonstrate that the
abnormal responsivenesst o G-CSF in canine cyclichematopoiesis is present in neutrophil precursors andis notassociated with demonstrable alterations in the number, binding
affinity, or overall size of the G - S F receptor in neutrophils,
or with defective tyrosine phosphorylation of p80. These
data suggestthat cyclic hematopoiesisis caused by a defect
in the G-CSF signal transduction pathway at a point distal
to G-CSF receptor binding that does not involve the early
biochemical eventsleadingt o p80 tyrosine phosphorylation.
0 7994 by The American Society of Hematology.
C
determinations. Blood samples for neutrophil isolations required between 50 and 120 mL and were timed in grey collies to maximize
the neutrophil yield.
In vitro colony formation assays. In vitro BM colony formation
was used to assess the potential responsiveness of canine marrow
cells to G-CSF. Assays were performed as previously de~cribed'~,'~
using normal and grey collie BM. BM was aspirated into heparin,
diluted, and subjected to Ficoll-Paque (Pharmacia, Piscataway, NJ)
density gradient centrifugation. To reduce background growth factor
production by cells in the marrow, two cycles of adherence to the
surface of plastic flasks (T-75; Falcon, Lincoln Park, NJ) for 1 hour
at 37°C were performed. The mononuclear cells were plated at 1 X
lo5 cellslmL in the presence of 10% fetal calf serum, 5% normal
dog serum, and varying concentrations of exogenous G-CSF. Recombinant human and canine G-CSF (rhG-CSF and rcG-CSF) were
kindly provided by Dr Lawrence Souza (Amgen, Thousand Oaks,
YCLIC HEMATOPOIESIS is a rare disorder in humans
and grey collie dogs characterized by regular cyclical
oscillations in blood counts and periodic severe neutropenia
frequently complicated by bacterial infection^."^ Although
the mechanisms underlying this disorder are unknown, recent clinical trials have shown that supraphysiologic doses of
granulocyte colony-stimulating factor (G-CSF) ameliorate
neutropenia in humans and eliminate cyclical hematopoiesis
in dogs with this disorder.""*
Abnormal responsiveness to G-CSF may be an important
pathophysiologic mechanism underlying congenital cyclic
hematopoiesis. We previously reported that abnormally increased concentrations in vitro of G-CSF and granulocytemacrophage-CSF (GM-CSF) but not interleukin-3 (IL-3)
were required to stimulate maximal granulocytic colony formation by bone marrow (BM) cell populations from patients
with congenital cyclic ne~tr0penia.l~
The requirement for
increased concentrations of growth factor could be demonstrated at the level of CD34' BM progenitor cells and was
most striking with G-CSF. These studies suggest that the
abnormality causing cyclic hematopoiesis in humans with
cyclic neutropenia lies in growth factor receptor binding or
in the postreceptor signal transduction system that drives
granulocytopoiesis. To investigate this hypothesis in the grey
collie model, we examined BM mononuclear cells and neutrophils from grey collie dogs to determine whether G-CSF
responsiveness and G-CSF receptor expression were abnormal.
MATERIALS AND METHODS
Dogs and blood sampling. Grey collie dogs (ages 1 to 4 years)
and normal mongrel dogs were housed in American Association
for Accreditation of Laboratory Animal Care (AAALAC)-accredited
animal care facilities as previously described.'"16 Daily blood counts
were performed between 7 AM and 9 AM, including a white blood
cell count with differential, hematocrit, reticulocyte, and platelet
Blood, Vol 84, No 3 (August l ) , 1994: pp 789-794
From the Division of Bone Marrow Transplantation, Department
of Internal Medicine and the Arthur G. James Cancer Hospital and
Research Institute of The Ohio State University, Columbus, OH; the
Division of Hematology, Department of Medicine of the University
of Washington and the Providence Medical Center, Seattle, WA;
the Division of Hematology and Medical Oncology, Oregon Health
Sciences University, Portland, OR; andthe Divisions of Cellular
and Molecular Biology and Tumor Immunologyof the Dana-Farber
Cancer Institute, Boston, MA.
Submitted July 2, 1993; accepted March 28, 1994.
Supported by National Institutes of Health Grants No. HL.01812,
DK44194, CA01422, CA36167, and AM18951.
Address reprint requests to Belinda R. Avalos, MD, Division of
Bone Marrow Transplantation, 303 E Doan Hall, 410 W Tenth Ave.
The Ohio State University, Columbus, OH 43210.
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 1994 by The American Society of Hematology.
0006-4971/94/8403-00I9$3.00/0
789
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790
CA). Colonies (greater than 50 cells) were counted at 8 to 10 days
("total colonies"). As a positive control, phytohemagglutinin-stimulated canine leukocyte conditioned medium (PHA-LCM) was routinely assayed as an estimate of maximal colony-forming capacity
for each marrow specimen. Data are presented as percent of maximal
colony formation."
Isolation of neutrophils. Neutrophils were isolated from heparinized peripheral blood by ficoll-hypaque gradient centrifugation followed by sedimentation in 3% dextran sulfate as previously described.I7 Cells (95% to 98% neutrophils) were washed once with
phosphate-buffered saline (PBS) then resuspended in binding buffer
consisting of Iscove's modified Dulbecco's medium (IMDM) containing 25 mmol/L HEPES and 4 mg/mL bovine serum albumin
(BSA), with the pH adjusted to 7.4.
Iodination of rhG-CSF. All receptor binding studies were performed with rhG-CSF. Five micrograms of the rhG-CSF analogue
Met-Lys-rhG-CSF (kindly provided by L. Souza and T. Boone, Amgen) was radioactively labeled using lactoperoxidase, glucose oxidase, and 1 mCi NaI (Amersham, Arlington Heights, IL) as previously described." The '*'I-labeled G-CSF was purified by G-50
sephadex gel filtration. This procedure resulted in specific radioactivities for '"I-G-CSF ranging from 1.7 to 4.6 X IO5cpm/ng as determined by self-displacement analyses.'7
G-CSF binding assay. Neutrophils (2.0 to 5.0 X 10') were incubated for 3 hours at 4°Cin binding buffer containing varying concentrations of "'I-G-CSF in the range of 0.01 to I O nmol/L in the
presence or absence of a 100-fold excess of unlabeled G-CSF in a
final volume of 400 pL. After incubation, the cells were resuspended,
transferred onto a 0.75-mL ice-cold mixture of 75% calf serum in
binding buffer and then centrifuged. The supernatants were aspirated
and the cell pellets were counted in a gamma counter. All binding
experiments were performed using duplicate incubations. Specific
binding was defined as the amount of binding competed by a 100fold excess of unlabeled G-CSF. Equilibrium binding data were
analyzed according to the method of Scatchard" and by weighted
nonlinear least-squares curve fitting as described by Munson and
Rodhard." Objective statistical criteria (F test, extra-sum squares
principle) were used to evaluate goodness of fit and for discriminating between models."
Afiniry labeling and immunoprecipitation of '*'I-G-CSF ligand
receptor complexes from dog neutrophils. A total of 5 X 10' neutrophils in 1.0 mL binding buffer was incubated for 3 hours at 4°C
with 500 pmol/L '*'I-G-CSF in the presence or absence of a 1 0 0 fold excess of unlabeled G-CSF. The cells were washed twice with
ice-cold PBS and resuspended in 1.O mL cold PBS, pH 8.3, to which
disuccinimidyl suberate (DSS) was added at a final concentration of
1 mmollL. The reaction was quenched after 30 minutes by the addition of 100 mmoVL Tris-HC1, pH 7.4, and the samples were washed
twice with cold PBS. To reduce background nonspecific binding
and uncross-linked '*'I-G-CSF, the pellets were resuspended in 70
mmol/L sodium acetate and 50 mmol/L NaCl, pH 4.0, for 10 minutes
at 0°C. The samples were washed twice with cold PBS and gently
rocked for 20 minutes at4°C in 1.0 mL lysis buffer consisting of
PBS with 1 % Triton, 2 mmol/L phenylmethylsulfonyl fluoride
(PMSF; Sigma, St Louis, MO), 10 pmol/L pepstatin (Sigma), 1
mg/mL aprotinin (Sigma), 2 mmollL EDTA, 10 p m o E leupeptin
(Sigma), and 1 mmol/L diisopropyl-fluorophosphate(DFP; Sigma).
The supernatants from spun samples were incubated overnight at 4°C
with 30 pL of rabbit antihuman G-CSF antisera that was generated
in our laboratory, then 12 hours later incubated with 10% washed
staphylococcal A protein (Pansorbin; Calbiochem, La Jolla, CA) for
4 hours at 4°C. The spun samples were washed three times in lysis
buffer and then resuspended in sodium dodecyl sulfate (SDS) sample
buffer containing 5% 2-mercaptoethanol. Aliquots of the supernatants were applied to a 7.5% SDS-polyacrylamide gel. The dried gel
AVALOS ET AL
was autoradiographed for 2 weeks at-70°C with an intensifying
screen.
Isolation of RNA and Northern analysis. Total RNA was isolated
by acid guanidinium thiocyanate-phenol-chloroform extraction."'
Ten-microgram total cellular RNA samples were fractionated by
electrophoresis through a 1% agarose formaldehyde gel, transferred
to nylon membranes, and hybridized to a minimum of 10' cpm/mL
"P-labeled probe for the human G-CSF receptor. A probe for the
human G-CSF receptor was prepared by Xba I digestion of the 2.5kb Bgl 2 bounded insert of the human G-CSF receptor cDNA clone
D7*' (kindly provided by Alf Larsen, Immunex, Seattle, WA) ligated
into the Bam site of pBluescript I1 Sk and labeled with [CU-~*P] dATP
by random oligonucleotide priming. The filters were hybridized at
42°C for 16 hours, washed to a final stringency of 0.25X SSC, and
exposed to Kodak X-Omat films (Eastman Kodak, Rochester, NY).
Immunoblnting of cell lysates. Neutrophils were isolated as described above after which the cell pellet was washed twice with
Hanks' Balanced Salt Solution (GIBCO, Grand Island, NY). Red
blood cells were lysed by incubation for 30minutes at 4°C in ammonium chloride (8.29 g/L, pH 7.27). The neutrophil pellet was resuspended in RPM1 1640 (GIBCO) containing 0.5% BSA (fraction V:
Sigma) at 5 X 10' cellslml. Neutrophils were pretreated with 1
mmol/L DFP for 30 minutes at 4°C to inhibit proteases before CCSF stimulation. Cells were then warmed to 37°C and treated with
varying concentrations of G-CSF from 0.01 to 10 ng/mL for 15
minutes. After stimulation, cells were washed in PBS and lysed in
lysis buffer (20 mmoVL Tris-HCI, pH 8.0, 150 mmol/L NaCI, IO%
glycerol, 1% NP-40), containing 1 mmol/L PMSF, 0.15 U/mL aprotinin, IO pg/mL leupeptin, and 1 mmoVL sodium orthovanadate
(Sigma) at 4 T for 20 minutes at a concentration of 10' cells/mL.
Insoluble material was removed by centrifugation at4°C for I O
minutes at 10,000g. Whole cell lysates (5 X 10' cells) were mixed
I : l with 2X SDS sample buffer containing 2-mercaptoethanol and
heated at 95°C for 3 minutes before one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with 7.5% polyacrylamide.
After electrophoresis, proteins were electrophoretically transferred
to 0.2 p nitrocellulose (Schleicher & Schuell, Keene, NH) in a buffer
containing 25 mmol/L Tris, 192 mmoVL glycine, and 20% methanol
at 0.4 Amp for 4 hours at 4°C. Residual binding sites were blocked
by incubation in TBS (10 mmoVL Tris, pH 8.0, 150 mmol/L NaCI)
containing 2% gelatin for 60 minutes at 25°C. The blots were incuhated overnight at room temperature with the antiphosphotyrosine
antibody 4G10 (1.5 pg/mL) in TBST (TBS with 0.05% Tween 20).
The 4GI0 antibody was generated using phosphotyramine as the
immunogen and antibody reactions were detected as described."."
Prestained molecular weight markers (BRL, Gaithersburg, MD) were
also loaded onto the gel.
RESULTS
In vitro colony growth. BM mononuclear cells from grey
collie dogs required sevenfold higher concentrations of GCSF to achieve half-maximal colony growth than cells from
normal dogs (56 pmol/L v 8 pmol/L), as shown in Fig 1 .
Similar dose-response curves wereobtained using either
rcG-CSF or rhG-CSF and were therefore pooled for presentation.
Receptor
expression
studies.
Neutrophils
from
three
grey collie and three normal dogs were examined in a total
of eight separate experiments for G-CSF receptor expression.
Representativebinding curves andScatchard analyses are
shown in Fig 2. Neutrophilsfromnormal dogs express a
single class of high-affinity receptors with 56 to 446 binding
sites per cell and a kd of 28 to 206 pmol/L (n = 4, Fig
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791
G-CSF RECEPTOR IN CANINE CYCLIC HEMATOPOIESIS
transcripts migrated with a slightly slower mobility than the
human neutrophil transcript.
Antiphosphotyrosine immunobbts. Neutrophils from
both normal and grey collie dogs were stimulated with
G-CSF at 0.01, 0.1, 1.0, and 10 ng/mL and evaluated by
immunoblotting with an antiphosphotyrosine antibody to determine whether the pattern of tyrosine phosphorylation induced by G-CSF in grey collie cells differed from that observed in normal dog cells. Rapid and prominent tyrosine
2om
1
00
100
200
300
400
rG-CSF (PM)
Fig 1. In vitrocolony assays. Nonadherent BM mononuclear cells
and grey collies (0)were plated at 1 x 10'
from normal dogs (0)
cells/mL in NCTC 109 medium containing 10% FCS, 5% normal dog
serum, and varying concentrations of rcG-CSF or rhG-CSF. Colonies
(>50 cells) were counted at 10days. The percentage of maximal
colony growth in each individual experiment is plottedas a function
of G-CSFconcentration. Data presented are derived
from the mean of
triplicate samples in each experiment,with a total of five experiments
from normal dogs and four experiments in grey collies. Error bars
represent +l
SD.
L
0
0
200
400
BOUND
600
(Dn)
800
1000
125
2A). Similar receptor numbers for G-CSF with a comparable
dissociation constant were observed on neutrophils from
grey collies with 78 to 199 receptors per cell and a kd of 84
to 195 pmol/L (n = 4, Fig 2B). G-CSF receptor expression
was also analyzed in one grey collie dog before and during
treatment with rcG-CSF. Before initiation of G-CSF therapy,
the number of G-CSF receptors on neutrophils from this dog
was 85 per cell and, on a repeat determination 18 months
later while on G-CSF, 86 per cell (data not shown).
Cross-linkingstudies.
Experiments were performed to
determine the size of the G-CSF binding proteins on neutrophils from normal and grey collie dogs. As shown in Fig 3,
when neutrophils from normal dogs and grey collies were
incubated with'251-labeledG-CSF,then
covalently crosslinked with DSS, immunoprecipitated with polyclonal antiG-CSF antibody, and analyzed by SDS-PAGE under reducing conditions, an identical major labeled band witha molecular weight of 139 kD was observed (see arrow). This band
was completely abolished on both normal and grey collie
neutrophils when a 100-fold excess of unlabeled G-CSF was
present during the incubation. Because the molecular weight
of G-CSF is 19 k D , the calculated molecular weight of the
G-CSF binding protein on normal and grey collie neutrophils
is approximately 120 M). Similar results were obtained and
confirmed by densitometry using BM mononuclear cells
from normal and grey collie dogs (data not shown).
Northern analysis. G-CSF receptor expression in normal
and grey collie cells was also examined by Northern blot
analysis. As shown in Fig 4, G-CSF receptor mRNA transcripts of identical size were detected in normal and grey
collie neutrophils. The canine neutrophil G-CSF receptor
5000
-
2000
-
l000
noun0
(Dm
0
0
400
200
125
I-G-CSF
600
800
1
1000
(pH)
Fig 2. Saturation binding curve for '%abeled G-CSF and Scatchard analyses. Binding of '%labeled recombinant G-CSF to neutrophils from a normal dog (A) and greycollie (B) is shown with Scatchard transformations of the data in the inset. Neutrophils (5 x 10'
cells) were incubatedfor 3 hours at 4°C with varying concentrations
of '%G-CSF in the absence or presence ofa 100-fold excess of unlabeled G-CSF. Specific binding shown on the vertical axis is the
amount of binding competed by excaw unlabeled G-CSF. Binding
parameters were
determinedby Scatchard analysis
of the data points
from the saturation binding curve using the UGAND program.In this
representative experiment, the calculated number of binding sites
on normal dog cells was 65 per cell with a Kd of84 pmollL, whereas
103 sites per cells with a Kd of 112 pmollL were detected on grey
collie cells.
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AVALOS ET AL
792
A
200
B
C
NI
D
-
-
.01
.l
1
10
-
GC
.Q1 .l l
10
201
-b
Q
97.4 -
f
p80
6
69
45
46
30
-
Ir
Fig 3, Cross-linking of ~
z
s G-CSF.
~
~z~
s~G - C
~~S ~
was
-~
covalently cross.linked to normal (A and B) and grey collie (c and
neutrophils (5 x l o cells)
~
as described in Materials and Methods in
the absence (A and c) and presence
and D) of
unlabeled
G-CSF. Migration of
weight markers is shown on the
left.
Fig 5. Dose-response of tyrosinephosphorylation after G-CSF
stimulation. Normal (NI) or gray collie (GC) dog neutrophils were
prepared as described in Materials andMethods. Lysates of unstimulated neutrophils I-) or neutrophils stimulated with the indicated
amounts of G-CSF in nanograms per milliliter were prepared. Proteins
were
separated
on
a 7.5%
SDS-polyacrylamide gel and immu~~
~~
~~
~
~
~
~
noblotted witha monoclonal antiphosphotyrosineantibody. The migration of an 80-kD proteinthat represents themajorinducible
tyrosine phosphorylated protein marked
is
with an arrow onthe right
and the migration of molecular weight markers is indicated on the
left of the panel.
I . ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~
phosphorylation of a protein of relative m o ~ e c u ~weight
ar
of
80 kD was Seen i n both
and grey collie neutrophils
( ~ 5 , iarrow)
~ and was most apparent at
concentrations of I ng/mL
and
I O ng/mL.
DISCUSSION
Cyclichematopoiesis in grey collie dogs is a stem cell
disease in which abnormal regulation of hematopoietic cell
production in the BM causes cyclicfluctuations of blood
cell counts.’.‘’ The canine disease is a close analogue of the
human disorder.’ Clinical trials in humans with this disorder
have demonstrated the requirement for supraphysiologic
doses of G-CSF to ameliorate neutropenia.”,”,~~
In grey collie dogs, neither IL-3 nor GM-CSFaltered cyclic fluctuations
in blood counts. whereas only high doses of G-CSF obliterated all of the cyclic fluctuations.” In humans with congenital cyclic neutropenia. abnormally increased concentrations
Canine
Human
A B C D E
3.7 kb -
F G H I
Fig 4. Northern blot analysis of G-CSF receptor
transcripts. Total RNA (10 pg) was hybridized to a
human G-CSF receptor cDNA probe prepared as described in Materials and Methods. Shown are samples isolated from (A) human
peripheral blood mononuclear cells, (B and C) human neutrophils fromt w o
different donors, (D) grey collie mononuclear cells,
(E) grey collie neutrophils, ( F ) normal dog mononuclear cells, and (G through 1) normal dog neutrophils
from three differentdogs.
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793
G-CSF RECEPTOR IN CANINE CYCLIC HEMATOPOIESIS
of G-CSF are required to stimulate in vitro granulocytic
colony formation by BM progenitor ~ e 1 l s .On
l ~ the basis of
these observations, a defect in G-CSF signal transduction
andor receptor expression has been implicated in the pathogenesis of cyclic hematopoiesis. In the present study, we
characterized the responsiveness of neutrophil precursors to
G-CSF in vitro and G-CSF receptor expression on mature
neutrophils to determine whether they are abnormal in the
model disorder in grey collies.
BM mononuclear cells from grey collies required abnormally high concentrations of G-CSF in vitro to achieve halfmaximal colony growth compared with cells from normal
dogs. This finding in vitro as well as our previous observations" in vivo that pharmacologic but not physiologic doses
of G-CSF eliminated cyclic oscillations in blood counts in
grey collies suggested to us that grey collies might express
reduced numbers of G-CSF receptors or receptors with an
altered affinity for G-CSF. We therefore examined binding
of IzI-labeled G-CSF to neutrophils from grey collies and
normal dogs. Scatchard analyses of the equilibrium binding
data were consistent with expression of a single class of
high-affinity receptors on both normal and grey collie cells
with similar receptor numbers and binding affinities. No variation in receptor numbers or affinity was observed in the
one dog studied during G-CSF treatment. The number and
affinity of G-CSF receptors on canine neutrophils is similar
to that previously reported for human neutrophils.'7.26-28
In this study, we also characterized the protein that binds
G-CSF on grey collie neutrophils. A binding protein of approximately 120 kD was identified on neutrophils from both
normal and grey collie dogs. No abnormal G-CSF receptor
mRNA transcripts were detected in grey collie neutrophils
by Northern analysis. These structural data suggested no
large defect in the G-CSF receptor itself.
Recently, Druker et a129reported rapid tyrosine phosphorylation of p80+"' in human neutrophils treated with G-CSF.
Because this is one of the first biochemical events in the
signal transduction pathway activated in neutrophils after
G-CSF stimulation, we investigated the patterns of protein
phosphorylation in normal and grey collie neutrophils after
treatment with G-CSF to determine whether G-CSF receptor
function in signal transduction is defective in grey collie
cells. A similar dose-response to G-CSF was observed. GCSF rapidly induced prominent tyrosine phosphorylation of
an 80-kD protein, suggesting that this part of the pathway
in G-CSF-mediated signal transduction is intact ingrey
collie cells.
The data presented here complement the previous observations by Kyas et al,24 who reported normal numbers of GCSF binding sites on neutrophils from humans with cyclic
neutropenia. In their study, no information was provided on
the size of the receptor protein or size of the mRNA transcripts of G-CSF receptors. In the present study, using the
canine model, we show that the abnormal response to GCSF in cyclic hematopoiesis is not caused by alterations in
the number or binding affinity of the G-CSF receptor expressed on neutrophils. However, it is possible that G-CSF
receptor expression on neutrophils differs from receptor expression on precursor cells and that the level of expression
or properties of the G-CSF receptor on grey collie precursor
cells are abnormal. Also, the size of the G-CSF receptor
protein and mRNA transcripts are not demonstrably altered
in cyclic hematopoiesis. We cannot exclude the possibility
of a small deletion or point mutation in the G-CSF receptor,
or expression of different G-CSF receptor molecules arising
from alternative splicing mechanisms, which would not be
detected by the methods used in our studies. Detection of
such a defect would require sequence data on the G-CSF
receptor gene in grey collie dogs. However, a sensitive functional assay of the receptor shows that one of the early signal
transduction pathways activated by G-CSF is clearly intact
in grey collie cells.
Taken together, our results show thatthe abnormal responsiveness to G-CSF in canine cyclic hematopoiesis is not
caused by gross alterations in the number, binding affinity,
or size of the neutrophil G-CSF receptor, or by abnormal
tyrosine phosphorylation of an 80-kD protein that behaves
like canine c-rel. These data suggest that the underlying
defect in cyclic hematopoiesis is in the G-CSF signal transduction pathway at a point distal to the G-CSF receptor.
ACKNOWLEDGMENT
We are grateful to Dr David C. Dale for providing us with the
opportunity to study the grey collie dogs. We also thank Norma
Haas for preparation of the manuscript.
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2. Dale DC, Ward SB, Kimball HR, WolffSM: Studies on neutrophil production and turnover in grey collie dogs with cyclic neutropenia. J Clin Invest 51:2190, 1972
3. Dale DC, Alling DW, Wolff SM: Cyclic hematopoiesis: The
mechanism of cyclic neutropenia in grey collie dogs. J Clin Invest
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4. Hammond WP, Dale DC: Cyclic hematopoiesis in grey collie
dogs, in Gershwin ME, Merchant B (eds): Immunologic Defects in
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5 . Jones JB, Lange RD: Cyclic hematopoiesis: Animal models.
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6. Guerry D, Dale DC, Omine M, Perry A, Wolff SM: Periodic
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7. Wright DG, Dale DC, Fauce AS, Wolff SM: Human cyclic
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10. Lothrop CD, Warren DJ, Souza LM, Jones JB, Moore MAS:
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12. Hammond WP, Price TH, Souza LM, Dale DC: Treatment
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