1. Art and Diseño

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Plasma/Serum Levels of flt3 Ligand Are Low in Normal Individuals
and Highly Elevated in Patients With Fanconi Anemia and Acquired
Aplastic Anemia
By Stewart D. Lyman, Michelle Seaberg, Roberta Hanna, JoDee Zappone, Ken Brasel, Janis L. Abkowitz,
Josef T. Prchal, John C. Schultz, and Nasrollah T. Shahidi
The fltt ligand is growth
a
factor that stimulates the proliferation of hematopoieticprogenitor andstem cells. We establishedasensitiveenzyme-linkedimmunosorbent
assay
(ELISA)to measurethe concentrationof flt3 ligand in plasma
or serum from normal individuals,as well as in patients with
hematopoietic disorders.Concentrationsof flt3 ligand in
plasma or serum from normal individuals were quite low:
only 12%(7 of 60)of normal individuals hadflt3 ligand levels
above 100 pg/mL (the limit of detection). In contrast, 86%
(19 of 22) of samplesfrom patients with Fanconi anemia and
100% (eight of eight) of samplesfrom patients with acquired
aplastic anemia had plasma or serum levels above 100 pg/
T
HE flt3 ligand is a member of a small group of cytokines
that affect the growth, survival, and/or differentiation
of hematopoietic cells through the activation of specific tyrosine kinase receptors.' Other members of this family include
Steel factor (also known as mast cell growth factor, c-kit
ligand, and stem cell factor') and colony-stimulating factor
1 (CSF-l), which is also known as macrophage colony-stimdating f a ~ t o rAll
. ~ of these proteins have a similar size and
structure and contain conserved cysteine residues that are
believed to form intramolecular disulfide bonds that give the
proteins their three-dimensional ~hape.4.~
Comparison of the
mouse and human flt3 ligand genomic loci5" with the genomic loci for Steel factor6 and CSF-1' led us to suggest that
all three of these hematopoietic growth factors are derived
from duplication of an ancestral gene.
The primary role of flt3 ligand in the hematopoietic system
appears to be the maintenance and proliferation of hematopoietic stem and progenitor ~ e l l s . ' ' ~By
~ ~itself,
~ ~ ~ flt3 ligand
does not have strong stimulatory effects on hematopoietic
cells, but it synergizes well with a number of other hematopoietic growth factor^.^.^,^"'
There are several mechanisms by which soluble flt3 ligand
may be generated in vivo. The primary translation product
of the flt3 ligand gene appears to be a transmembrane protein
that can undergo proteolytic cleavage to generate a soluble,
biologically active form of the protein!.'.'
In addition, alternative splicing of flt3 ligand mRNAs can take place to generate other soluble isoforms of the protein that are also biologicallyactive."" This can occur by either splicing out the
transmembrane region' or insertion of a sixth exon that introduces a stop codon into the extracellular domain.'*
As part of our efforts to define the role of this protein
in normal human hematopoiesis, we established a sensitive
enzyme-linked immunosorbent assay (ELISA) for measuring
flt3 ligand levels in human plasma and serum. We now report
that flt3 ligand levels are elevated in patients with Fanconi
anemia and acquired aplastic anemia, but not inpatients with
several other hematopoietic disorders involving single cell
lineages. This finding supports the notion that flt3 ligand is
involved in the regulation of hematopoietic stem cells and
suggests that flt3 ligand levels rise in response to a need for
stem cell expansion.
Blood, Vol 86,No 1 1 (December l), 1995:pp 4091-4096
mL. Mean plasma or serum concentrations (calculated by
assigning a value of 0 pg/mL to any sample reading below
the level of detection) were as follows: normal volunteers,
14 pg/mL; patients with Fanconi anemia, 1,331 pg/mL; and
patients with acquired aplastic anemia,460 pg/mL. Concentrations of flt3 ligand in blood are, therefore, specifically elevated to a level that may be physiologicallyrelevant in
hematopoieticdisorders with a suspectedstem cell component. The elevated flt3 ligand concentrationsin these individuals may be part of a compensatory hematopoietic response to boost the level of progenitor cells.
0 1995 by The American Societyof Hematology.
MATERIALSANDMETHODS
Generation of antibodiesto human fit3 ligand. Antibodies to
human flt3 ligand were made against recombinant FLAG yeastderived flt3 ligand: as outlined below. The term "FLAG" designates the presence of an eight-amino acid sequence at the N-terminus
of the protein that facilitates its purification via anti-FLAG antibodies.''
Human j t 3 ligand polyclonalantibodyproduction.
A New
Zealand white rabbit was immunized subcutaneously with 25 pg
FLAG yeast-derived human flt3 ligand extracellular domain (FLAGhuman flt3 ligand) to generate polyclonal antisera. This antiserum
was designated P1 (polyclonal 1).
Production of anti-human j t 3 ligand monoclonal antibody.
Lewis rats were injected subcutaneously with 10 pg FLAG yeastderived human flt3 ligand extracellular domain in Freund's complete
adjuvant. After two immunizations, one rat had immunoblot titers
greater than 1:6,400, with no reactivity to an irrelevant FLAG protein. That rat was given an intrasplenic immunization of 2 pg FLAG
yeast-derived human flt3 ligand in saline, and 3 days later, the spleen
cells were fused with the NSl myeloma cell line using polyethylene
glycol. Fusion plates and cloning plates were screened by an antibody capture plate assay using biotinylated FLAG yeast-derived
human flt3 ligand and by fluorescence-activated cell sorting (FACs)
analysis using CV1 cells transfected with human flt3 ligand cDNA.
Antibodies positive by FACS were also tested by ELISA against an
irrelevant FLAG protein to be sure that they were not FLAG-reactive. Monoclonal antibody M5 (IgG2a isotype) tested positive in
these assays and in an immunoprecipitation assay using flt3 ligand
cDNA-transfected CV 1 cells. Preliminary data indicate that the M5
From Immunex Corporation, Seattle, WA;the Department of Hematology,University of Washington, Seattle, WA; the Division of
Hematology/Oncology, Department of Medicine, university of Alabama-Birmingham, Birmingham, AL; and the Division of Pediatric
Hematology/Oncology. Departmentof Pediatrics, Universityof Wisconsin, Madison, WI.
Submitted June I, 1995; accepted July 21, 1995.
Address reprint requests to Stewart D. Lyman, PhD, Immunex
Corporation, 51 University St, Seattle, WA 98101.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore behereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8611-0019$3.00/0
4091
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4092
antibody recognizes an epitope within the last 22 amino acids of the
extracellular domain of flt3 ligand (data not shown).
Humanjt3 ligand ELISA. Wells of polystyrene microtiter plates
(Maxisorp; NUNC, Roskilde, Denmark) were coated with 100 pL
of a 0.1 p g h L solution of ascites-produced humanflt3 ligandspecific monoclonal antibody M5 in 0.01 molL phosphate-buffered
saline (PBS), pH7.2,and
incubated at2°Cto
8°C overnight. A
Chinese hamster ovary (CH0)-derived human flt3 ligand standard
(Immunex Corp, Seattle, WA) andhuman sera were diluted in a
sample buffer comprised of PBS with 0.05% Tween-20 (PBST) with
an additional 0.5 mol/L NaCl and 5% normal rat serum. The human
flt3 ligand standard curve ranged from 1,600to 25 pg/mL in twofold
increments. Sera were tested at 1 :4, 1:8,I :16, and 1:32 dilutions in
duplicate. All reactants were dispensed in 100 p1 per well volumes,
and the wells were washed with PBST before the addition and incubation of each successive reactant. Standards and sera were incubated
for I houratroom
temperature (RT), followed by al-hour RT
incubation of a solution of rabbit anti-human flt3 ligand polyclonal
PI, followed by a l-hour RT incubation of a solution of peroxidaseconjugated donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Color was developed with 3,3‘,5,5’-tetramethylbenzidine (TMB) peroxidase substratekhromogen solution
(Kirkegaard & Perry Laboratories. Gaithersburg, MD) for 10 minutes
at RT. The reaction was stopped with 1 m o m H,PO,, and optical
densities were determined at a wavelengthof 450 nm. DeltaSoft
microplate analysis software (BioMetallics, Princeton, NJ) was used
to fit the standard curve by a four-parameter logistic model and to
estimate sample concentrations by interpolation from the fittedcurve.
Standards used for At3 ligand (CHO-derived or yeast-derived) were
amino acid-analyzed to determine protein concentration. The ELISAS were performed blinded, without knowledge of the hematopoietic status of the sample donors.
Inhibition of detection of humanfIt3 ligand by soluble humanfIf.3
receptor (huFlt3-Fc). A soluble version of the human flt3 receptor
was constructed using the same method that was used to engineer
a soluble version of the murine flt3 r e ~ e p t o rThe
. ~ extracellular domain of the receptor is fused to the Fc region of human IgG, and
the protein is referred to as huFlt3-Fc. Samples containing flt3 ligand
and huFlt3-Fc in molar ratios ranging from 1:1,000 to 1:O.g were
prepared and preincubated at RT for 2 hours before analysis in the
ELISA. The flt3 ligand concentration was held constant at 800 pg/
mL, whilethe concentration of huFlt3-Fc was varied. A sample
offlt3 ligand without huFlt3-Fc served as a reference control for
calculations of percent flt3 ligand detection.
fIr3 ligand bioassay. Bioactivity ofAt3 ligand in human plasma
samples was determined by measuring their capacity to stimulate
the proliferation of the murine WWF7 cell line in a [%-thymidine
incorporation assay, as previously described.” Recombinant, soluble
humanflt3ligandproduced
in C H 0 cells wasused as a positive
control in these bioassays and was spiked into pooled normal human
male serum (Sigma, St Louis, MO). A soluble form of the human
flt3 receptor (huFlt3-Fc fusion protein, see above) was used toinhibit
flt3 ligand bioactivity and thereby demonstrate the specificity of the
proliferative response. The final concentration of the FIt3-Fc fusion
protein in those assays where it was included was 2 pg/mL.
Collection of human blood samples. All blood samples were
collected in compliance with each institution’s guidelines for human
subjects. Blood samples were collected from normal volunteers after
obtaining informed consent. Samples from patients with hematologic
disorders were collected in the course of clinical evaluation. Plasma
samples were prepared by collecting blood in tubes containing heparinandthen removing the cells by centrifugation. Serum samples
were prepared by allowing the blood to clot and then removing the
clot by centrifugation. Total numbers of samples evaluated for each
group arereported below, withthe number of plasmahumber of
LYMAN ET AL
semm samples in parentheses: normal controls, 60 (49/11); Fanconi
anemia, 22 (22/0); aplastic anemia, eight (seven/one); pure red cell
aplasia, 13 (onell2); polycythemias, eight (none/eight); DiamondBlackfan anemia, five (twohhree); cy thalassemia (two-gene deletion), three (threehone); anemia of undetermined origin with intact
myelopoiesis, seven (sevenhone); idiopathic thrombocytopenia purpura, one (onehone). Plasma and serum samples were stored frozen
at -20°C or at -70°C before use.
A diagnosis of Fanconi anemia was established by the presence
of various congenital malformations and/or hematologic abnormalities and by the demonstration of an increased frequency of chromosomal breakage in the presence of diepoxybutane. Patients with acquired aplastic anemia fulfilled the following criteria: hemoglobin
level c 1 0 g/dL or hematocrit 530%, platelet count 5 2 0 X IOh/pL,
and granulocyte count 50.5 X 1031pL.The bone marrow cellularity
(ascertained by bone marrow biopsy) was less than 25% in all patients andwas devoid ofany neoplastic infiltration or significant
fibrosis. None of the patients with acquired aplastic anemia exhibited
congenital anomalies, growth retardation, or increased chromosomal
breakage in the presence of diepoxybutane.
Patients with pure red cell aplasia had severe anemia (hematocrit
less than 23%) and reticulocytopenia, while granulocyte and platelet
counts were normal. Hemoglobinized cells comprised less than 2%
of nucleated cells in the marrow aspirate. Patients with DiamondBlackfan anemia had marrow erythroid hypoplasia and either a high
mean corpuscular volume or fetal hemoglobin level. All patients
met standard diagnostic criteria.I4
Eight patients in our study had polycythemias, three ofwhom
had polycythemia rubra vera. These three subjects had documented
elevation of hemoglobin concentration with confirmed elevation of
red cell mass, as measured by [“‘Crl-labeling ofred cells using
standard methods. All subjects had elevated platelet counts. The
diagnosis of polycythemia vera was confirmed by demonstration of
a normal or low serum erythropoietin level, normal hemoglobin
oxygen dissociation-P50 determination, and erythropoietin-independent colonies in clonogenic assays of erythropoietin progenitors.
Three additional subjects had secondary congenital polycythemia,
normalP50,normal arterial blood gases, and stable, significantly
elevated erythropoietin levels. Polycythemia was confirmed by red
cell blood volume measured by [”Cr] assay. Two other subjects had
primary congenital polycythemia with a low erythropoietin level,
increased sensitivity of erythroid progenitors to erythropoietin, and
normal white blood cells and platelets. These two unrelated patients
had different mutations ofthe erythropoietin receptor in its cytoplasmic domain, which was shown to result in the hypersensitivity
of erythroid progenitors to erythropoietin.
Statistical analysis. Wilcoxon two-sample tests were performed
pairwise to compare the various patient and control groups statistically.
RESULTS
Establishment of the ELISA. Monoclonal and polyclonal
antibodies raised against human yeast-derived flt3 ligand
were used to establish a sandwich ELISA. Although the
assay was developed using antibodies directed against flt3
ligand made in yeast, no difference was seen instandard
curves calibrated using either yeast-derived flt3 ligand or flt3
ligand produced by CH0 cells (Fig 1). We were unable to use
the native flt3 ligand protein as an ELISA standard because it
has not been purified from human blood, and we have not
determined the exact C-terminus of the native protein. However, there appeared to be no difficulties in measuring this
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4093
SERUM LEVELS OF flDL ARE ELEVATED IN FNAA
n
E
P
c
0
*
v)
1.51-
0.5-
Fig 1. Standard curve of yeast-derived and CHO-derived flt3 ligand in the ELISA. The graph shows the standard curve used to
determine plasma or serum levels of flt3 ligand. Recombinant soluble
read
human flt3 ligand produced in either yeast (U)or CH0 cells (0)
out identically in this assay.
protein in plasmaherum, even though it appears to be present
at very low levels.
There have been no reports in the literature of soluble flt3
receptors in human blood. However, given the high serum
levels (mean, 324 ng/mL) reported for soluble c-kit receptor," we tested whether soluble human flt3 receptor could
inhibit the capacity of the ELISA to measure flt3 ligand.
This was accomplished using a fusion protein that links the
extracellular domain of the human flt3 receptor to the Ig
portion of human IgG. No inhibition of ligand binding was
seen until a sixfold molar ratio of receptor to ligand was
reached (Fig 2). Maximal inhibition of about 70% occurred
at approximately a 100-fold molar excess; increasing the
amount of soluble receptor to a 1,000-fold molar excess
produced no additional inhibition.
P l a s d s e r u m levels offlt3 ligand. In most normal individuals, the flt3 ligand plasmdserum concentration was less
than 100 pg/mL, which is the limit of detection of our ELISA
(Fig 3). The highest level measured in a normal person was
only 152 pg/mL. Normal plasmdserum levels of flt3 ligand
are, therefore, significantly lower than the normal levels of
two related growth factors, CSF- 1I6.I7 and Steel factor,'' both
of which are normally found in the 1- to 8-ng/mL range.
Plasmaherum flt3 ligand levels measured in patients with
pure red cell aplasia (n = 12), a thalassemia (n = 3), Diamond-Blackfan anemia (n = 5), anemia of undetermined
origin (n = 7), or idiopathic thrombocytopenia purpura (n
= 1) were also quite low, with most of the samples being
below our level of detection. We also measured flt3 ligand
levels in serum from patients with various polycythemias (n
= 8) and found these to be in the normal range (Fig 3).
In contrast with these results, flt3 ligand plasma levels in
patients with either Fanconi or aplastic anemias were greatly
elevated (Fig 3). Whereas only12% (7 of 60) of normal
individuals had flt3 ligand levels above 100 pg/mL, 86% (19
of 22) of patients with Fanconi anemia and 100% (eight of
eight) of patients with acquired aplastic anemia had plasma
levels above this amount. We assigned a value of 0 pg/mL
to any sample that was below our ELISA level of detection
of 100 pg/mL to calculate mean plasmdserum levels. This
enabled us to calculate the following mean +- SD plasmal
serum flt3 ligand concentrations: normal individuals, 14 +39 pg/mL (median, 0 pg/mL; range, 0 to 152 pg/mL); Fanconi anemia, 1,331 2 2,350 pg/mL (median, 453 pg/mL;
range, 0 to 10,815 pg/mL); and aplastic anemia, 460 2 187
pg/mL (median, 426 pg/mL; range, 223 to887 pg/mL).
Plasmdserum concentrations of flt3 ligand in the Fanconi
or aplastic anemia groups were significantly greater than
both the normal donor group ( P = .0001) and the pure red
cell aplasia (mean, 27 5 64 pg/mL; median, 0 pg/mL; range,
0 to 191 pg/mL) or polycythemia (mean, 15 2 41 pg/mL;
median, 0 pg/mL; range, 0 to 123 pg/mL) groups (P =
.Owl). Thus, flt3 ligand levels are substantially elevated in
patients with these two stem cell-based anemias compared
with normal individuals or those with several other hematopoietic disorders. Analysis of the levels of flt3ligand in
patients with Fanconi anemia as a function of age, transfusion status, hemoglobin level, or disease severity showed
that there was neither a positive nor negative correlation
between these parameters (data not shown). The variations
in flt3 ligand levels are much greater in the samples from
patients with Fanconi anemia thanin those from patients
with aplastic anemia. We believe that most of the increased
100-
80 60-
40 l
20 -
1
0
0.5 1
10
100
1000
Ratio Flt3-FdFlt3 Ligand
Fig 2. Effect of soluble flt3 receptor on detection of human flt3
ligand by EUSA.Human ftt3 ligand ( 8 0 0 pglmLI and varying amounts
of soluble human fk3 receptor (a humanFlt3-F~
fusion protein) were
mixed in molar ratios ranging from 1:0.8to 1:l.OOO and incubated at
room temperature for 2 hours before analysis in theELISA. Asample
of human fk3ligand without FM-Fcserved as a darence control for
calculations of percent human flt3 ligand detection.
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4094
LYMAN ET AL
a
a
a
a
E
B
a
e
v
2m
1000
a
A
.0,
9
i
i
!
A
a
t
f
a
100
1
a
53
3
"
01
v
0
Fanconi
Normal
Acquired
Anemia
1331 Wml
14pg/ml
Aplastic
Anemia
480 pg/ml
11
7
Pure
Polycythemias
Red Cell
Aplasia
27 pg/ml
variability is likely due to the sample size, which is almost
three times larger for the Fanconi group (22 patients) relative
to the aplastic anemia group (8 patients).
Plasma samples from the three Fanconi anemia patients
with flt3 ligand concentrations above 3,500 pg/mL (Fig 3)
were tested in a proliferation assay to determine if the flt3
ligand measured in the ELISA was biologically active. All
three of these Fanconi anemia plasma samples stimulated
the proliferation of WWF7 cells, and this activity was specifically inhibited by the addition of soluble flt3 receptor
(Fig 4). In contrast, plasma samples from the three patients
-g
400
-
c"
300
-
1
10
100
1lDilution
pg/ml
1s
,6
Detection
Limit
aThalasaemla 3
/7]
AnemiaOther
Diamond-Blackfan
Anemia (5)
ITP (1)
Fig 3. Levels of flt3 ligand in
plasma or serum
from normal individuals and those
with various
hematopoietic
disorders.
The
numbers shown below the 100pglmL line are the numbers of
individuals whose serum levels
measured below the limit of detection in the ELISA. Mean flt3
ligand concentrations in plasma
andserumare shown beneath
eachgroup.Todetermine
this
0 pglmL was
number, a value of
given to all samples that read
out below the level of detection
in the ELISA."Anemia-Other"
refers to anemias of undetermined origin with intact myelopoiesis.
with Fanconi anemia that had no detectable flt3 ligand by
ELISA did not stimulate the proliferation of the WWF7 cells
(data not shown). The specific activity of endogenous flt3
ligand in the Fanconi plasma samples is very similar to the
specific activity of the recombinant flt3 ligand produced in
CH0 cells (data not shown). Whether the elevated levels of
flt3ligand in patients with Fanconi anemia and acquired
aplastic anemia are physiologically effective in stimulating
hematopoiesis cannot be experimentally resolved.
One of the patients with Fanconi anemia (patient E.M.)
received an HLA-matched cord blood transplant from a sib-
1000
1 /Dilution
Fig 4. Bioactivity of flt3 ligand in plasma from patients with Fanconi anemia.(AI Proliierativeresponse of the W
F7 cell line to recombinant.
CHO-derivedflt3 ligand that was spiked into normal human serum (Sigma). The highestconcentration of ftt3 ligand in the assay was 25 ngl
ml: serial dilutions are twofold. This activity was complotdy inhibited by the addition of 2 pglmL soluble flt3 receptor. (B) Proliferative
7 cells to plasma from three patients with Fanconi anemiain the absence or presence of2 pglmL FM-Fc. Platme levels of
response of M
flt3 ligand (determinedby EUSA) for these three patients were as follows: S.P., 10,815 pglml; L.K., 3,560 pg/mL; D.W., 4,755 pglmL. The Flt3Fe completely inhibited the flt3 ligand activity in plasma from all three patients.
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SERUM LEVELS OF flt3L AREELEVATED
IN FAJAA
4095
tion of a flt3 ligand-responsive cell line. However, these
levels of flt3 ligand would not be predicted to be high enough
to saturate flt3 receptors and, therefore, would be unlikely
to produce a maximal physiologic response. The level of flt3
ligand measured in plasmdserum from normal individuals
(generally less than 100 pg/mL) is significantly below the
amount required to see a proliferative response in vitro, suggesting that normal circulating levels of flt3 ligand are not
high enough to be physiologically active. However, our measurements of plasmdserum levels of flt3 ligand do not address the role cell surface flt3 ligand plays in hematopoiesis.
Soluble Steel factor has been shown to have different bio-O!
\-,
logic
effects in vivo compared with cell surface-expressed
1/1/88
1/1/89
1/1/90
1/1/91
1/1/92
1/1/93
1/1/94
1/1/95
Steel factoP0*21;
the same may be true for flt3 ligand as well.
Date
Several of the patients with Fanconi anemia that had elevated
Fig 5. Plasma levels of flt3 ligand in patient E.M. with Fanconi
plasma flt3 ligand concentrations also provided bone marrow
anemia before and after undergoinganHLA-matchedcordblood
aspirates. Levels of flt3 ligand in plasma and bone marrow
transplant.'s
were very similar (data not shown).
One complicating factor in attempting to determine circulating levels of flt3 ligand is the fact that there may be soluble
ling in an attempt to cure his pancytopenia." Multiple plasma
flt3 receptors present in serum that interfere with our ELISA.
samples collected both before and after the transplant
No reports of soluble flt3 receptor have been published to
showed that flt3 ligand levels were markedly elevated before
date, but levels of a soluble form of the c-kit receptor, which
the transplant and then declined after the transplant, until
is structurally related to flt3, have been reported to be exthey decreased below the level of detection (Fig 5). The
tremely high in human serum.15 The high levels of c-kit did
transplant was successful in curing the bone marrow failnot, however, prevent measurement of soluble Steel factor
ure.I9
in serum. We acknowledge that it is possible that our ELISA
is reflecting differences in the serum levels of flt3 receptor,
DISCUSSION
which may result in differences in free (ie, unbound) ligand
that can be measured in our assay.
The data presented here show that flt3 ligand levels in
One issue that we have not addressed is the mechanism
plasmdserum from humans are normally quite low, but are
responsible for generating both the normal and elevated levelevated to a high degree in acquired and constitutional
els of circulating, soluble flt3 ligand in human serum. Soluaplastic anemias. Our hypothesis is that the elevated flt3
ble flt3 ligand can be generated by either proteolytic cleavage
ligand levels in Fanconi anemia and acquired aplastic anemia
of a transmembrane protein or alternative splicing of
patients result from a physiologic attempt to compensate for
mRNAs."
The exact tissue source(s) that produces the soluan intrinsic deficiency in their stem cell compartment. In this
ble flt3 ligand detected in serum is unknown, and the putative
model, the hematopoietic system attempts to compensate for
protease that cleaves the transmembrane isofom of flt3 lia stem cell deficiency by inducing production of a factor,
flt3 ligand, that stimulates the proliferation of these ~ e l l s . 4 . ~ , ~gand has not been identified. Expression of this protease
does not appear to be widespread, because a number of cell
In contrast, flt3 ligand plasmdserum levels are not elevated
lines show surface expression of flt3 ligand, yetmedium
in pure red cell aplasia, Diamond-Blackfan anemia, anemias
conditioned by these cells contains no detectable flt3 ligand
of undetermined origin, and a thalassemia, because these
as determined by ELISA or bioassay.22The transmembrane
hematopoietic disorders are not at the level of the stem cell
isoforms of CSF-1 and Steel factor are also proteolytically
and instead are restricted primarily to the erythroid lineage.
cleaved to generate soluble, biologically active proteins, and
Polycythemia vera is a myeloproliferative disorder associthe protease(s) responsible for those reactions has also not
ated with the overproduction of cells from multiple hematobeen identified.
poietic lineages, primary the erythroid lineage. According to
A comparison of the levels of flt3 ligand and Steel factor
our hypothesis outlined above, patients with polycythemia
in normal individuals and those with hematopoietic disorders
vera would not be expected to have elevated flt3 ligand levels
reveals some striking differences. Levels of Steel factor (as
because they have no stem cell deficiency and, therefore, no
need to produce a factor that functions to produce more of
well as CSF-1) in normal human serum average several
these cells. Levels of flt3 ligand were not elevated in any of
nanograms per
much higher than the levels of
the polycythemia samples, including the three patients with
flt3 ligand reported here. Mean Steel factor serum levels
polycythemia vera (Fig 3), supporting our hypothesis that
were somewhat lower in patients with aplastic anemia comflt3 ligand blood levels are only elevated in disorders where
pared with normal individ~als,'~and the same is true for
there is a lack of stem or progenitor cells.
patients with Fanconi anemia (N.T.S. and J.C.S., unpubWe have shown in this report that the elevated levels of
lished observation, February 1995). Similarly, mean serum
soluble flt3 ligand observed in some of the Fanconi anemia
levels of Steel factor were about 25% lower than normal in
plasma samples are high enough to stimulate the proliferapatients with a variety of preleukemic disorders compared
't
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4096
LYMAN ET AL
with normal i n d i v i d ~ a l sSerum
. ~ ~ levels of Steel factor appeared to be somewhat elevated in only 2 of 15 patients with
Diamond-Blackfan anemiaz5(J.L.A., unpublished data, May
1995). Our central conclusion from these published studies
as well as our data presented here is that circulating levels of
flt3 ligand are significantly elevated in patients with Fanconi
anemia or acquired aplastic anemia, whereas serum levels
of Steel factor appear to be essentially normal or somewhat
decreased. These results underscore the fact that while both
flt3ligand and Steel factor stimulate the proliferation of
hematopoietic progenitor cells, there appear to be major differences in their biologic regulation.
ACKNOWLEDGMENT
We acknowledge Cheryl Koffley, Heidi Downey, and Sabine Escobar for characterizing the antibodies; Carlos Escobar for overseeing antibody production; Mike Widmer, Me1 Lebsack, and Douglas
Williams for critically reading the manuscript; and Abbe Sue Rubin
for the statistical analysis.
REFERENCES
1. Lyman SD, Williams DE: Biology and potential clinical applications of flt3 ligand. Curr Opin Hematol 2:177, 1995
2. Galli SJ, Zsebo KM, Geissler EN: The kit ligand, stem cell
factor. Adv Immunol 55:1, 1994
3. Roth P, Stanley ER: The biologyof CSF-l and its receptor.
Curr Top Microbiol Immunol 181:141, 1992
4. Lyman SD, James L,VandenBos T, de Vries P, BraselK,
Gliniak B, Hollingsworth LT, Picha KS, McKenna HJ, Splett RR,
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 86: 4091-4096
Plasma/serum levels of flt3 ligand are low in normal individuals and
highly elevated in patients with Fanconi anemia and acquired aplastic
anemia
SD Lyman, M Seaberg, R Hanna, J Zappone, K Brasel, JL Abkowitz, JT Prchal, JC Schultz and
NT Shahidi
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