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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 1967-1972
Interleukin-10 Inhibits Erythropoietin-Independent Growth of
Erythroid Bursts in Patients With Polycythemia Vera
By
Klaus Geissler,
Leopold Öhler,
Manuela Födinger,
Eva Kabrna,
Marietta Kollars,
Sonja Skoupy, and
Klaus Lechner
From the Division of Hematology of the 1st Medical Department, and
Department of Laboratory Medicine, University of Vienna, Vienna,
Austria.
 |
ABSTRACT |
In polycythemia vera (PV) erythroid colonies that grow in vitro in
the absence of exogenous erythropoietin (EPO) arise from the abnormal
clone that is responsible for overproduction of red blood cells.
Although the mechanism of autonomous formation of burst-forming
units-erythroid (BFU-E) is not fully understood, a spontaneous release
of growth regulatory molecules by PV cells and/or by accessory
cells is likely to be involved. Because of its cytokine synthesis
inhibiting action, interleukin-10 (IL-10) could be a potentially useful
molecule to modulate abnormal erythropoiesis in PV. We studied the
effect of recombinant human IL-10 on the EPO-independent growth of
erythroid bursts derived from peripheral blood mononuclear cells
(PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent,
and specific inhibitory effect on autonomous BFU-E formation. Ten
nanograms per milliliter of IL-10 significantly suppressed spontaneous
growth of erythroid colonies in methylcellulose in five of five PV
patients tested with a mean inhibition by 81% (range, 72-94). To
elucidate the possible mechanism of the inhibitory action of IL-10 we
further studied the effect of anticytokine antibodies on autonomous
BFU-E growth and the ability of exogenous cytokines to restore
IL-10-induced suppression of erythroid colony growth. Among a panel of
growth regulatory factors tested (granulocyte-macrophage
colony-stimulating factor [GM-CSF], IL-3, granulocyte
colony-stimulating factor, stem cell factor, and insulin-like growth
factor-1) GM-CSF was the only molecule for which both an inhibition of
spontaneous BFU-E formation by its respective antibody as well as a
significant restimulation of erythroid colonies in IL-10-treated
cultures by exogenous addition was found. Moreover, inhibition of
GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level.
Our data indicate that autonomous BFU-E growth in PV can be profoundly
inhibited by IL-10 and that this inhibitory effect seems to be at least
in part secondary to suppression of endogenous GM-CSF production.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
POLYCYTHEMIA VERA (PV) is a clonal
disease of the multipotential progenitor cell characterized by an
increased red blood cell mass and varying numbers of granulocytes and
platelets in the peripheral blood (PB). Whereas erythroid progenitors
from normal individuals require addition of exogenous erythropoietin
(EPO) to form erythroid colonies in vitro, a distinct population of erythroid progenitors from patients with PV can form hemoglobinized colonies in the absence of added EPO.1 Because such
spontaneous burst forming unit-erythroid (BFU-E) formation from PV
cells can still be observed under serum-free culture
conditions2 or in the presence of anti-EPO and
anti-EPO-receptor antibodies,2,3 minute amounts of EPO in
the culture system can be excluded as a possible cause of endogenous
colony formation. Growth of BFU-E without addition of exogenous growth
factors increases the possibility that PV cells secrete their own
growth factors and/or are stimulated by growth factors released
from accessory cells that might subsequently lead to hyperproliferation
of erythroid cells in vitro and possibly in vivo.
Interleukin-10 (IL-10) is a 35-kD protein, originally identified by
virtue of its ability to inhibit cytokine synthesis in T helper 1 clones.4,5 It is primarily produced by mononuclear cells
(MNCs)6 and possesses a wide range of activities on a number of cell types including B cells,7 T
cells,8 natural killer cells,9 mast
cells,10 neutrophils,11
eosinophils,12 and monocytes.13 The main
feature of this cytokine is a suppressive effect on cytokine
expression. Thus, IL-10 inhibits production of numerous cytokines in
lipopolysaccharide or interferon- -activated monocytes, such as
IL-1a, IL-1b, IL-6, IL-8, tumor necrosis factor- , granulocyte-
macrophage colony-stimulating factor (GM-CSF) and granulocyte
colony-stimulating factor (G-CSF).13,14
Because of its cytokine synthesis-inhibiting action, IL-10 could be a
potentially useful molecule to modulate hematopoiesis in conditions in
which the autocrine and/or paracrine secretion of cytokines
plays a significant role. In fact, we have shown that IL-10 markedly
suppresses the spontaneous formation of granulocyte-macrophage colonies
(CFU-GM) from normal PBMNCs in semisolid cultures.15 More
importantly, we showed a profound inhibitory effect of IL-10 on the
massively increased autonomous CFU-GM growth in patients with chronic
myelomonocytic leukemia (CMML), suggesting that IL-10 was a potential
therapeutic agent in this disease.16 Here we investigated
the effect of IL-10 on the spontaneous growth of erythroid colonies in
patients with PV. We found that IL-10 profoundly inhibits
EPO-independent BFU-E growth from such patients, at least in part
through suppression of spontaneous release of GM-CSF.
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MATERIALS AND METHODS |
Patients.
Five patients clinically diagnosed as having PV with the help of the PV
Study Group guidelines17 were used in this study. Clinical
and laboratory data of these patients are shown in
Table 1. All patients were being managed by
phlebotomy at the time of study and none of them had been previously
treated with cytostatic drugs or radioactive phosphorus.
Preparation of cells.
After informed consent, PB was collected into sterile tubes containing
EDTA. MNCs were isolated from PB of patients by Ficoll-Hypaque density
gradient centrifugation (density 1.077 g/mL, 400g for 40 minutes). The low-density cells were collected from the interface between density solution and plasma, washed twice, and resuspended in
Iscove`s modified Dulbecco`s medium (GIBCO, Paisley, Scotland).
Reagents.
Recombinant human IL-10 (rhIL-10; specific activity 1-2 × 106 U/mg) was kindly provided by
Schering-Plough Corp (Kenilworth, NJ) and rhGM-CSF and rhIL-3 by Sandoz
(Basel, Switzerland). RhG-CSF was purchased from British Biotechnology
(Oxan, UK) and rhEPO from Boehringer Mannheim (Vienna, Austria).
Recombinant human stem cell factor (rhSCF) and recombinant human
insulin-like growth factor-1 (rhIGF-1) were obtained from Pharma
Biotechnologie Hannover (Hannover, Germany). Antibodies directed
against GM-CSF, IL-3, G-CSF, and SCF were purchased from Genzyme
(Cambridge, MA), anti-IGF-1 from Serotec Ltd (Oxford, UK), and
anti-IL-10 from R&D Systems Europe Ltd (Abington, UK).
Colony assay.
PBMNCs were cultured in 0.9% methylcellulose, 30% fetal calf serum
(FCS; INLIFE, Wiener Neudorf, Austria), 10% bovine serum albumin
(Behring, Marburg, Germany), -thioglycerol (10-4 mol/L)
and Iscove`s modified Dulbecco`s medium with or without the addition
of cytokines or anticytokine antibodies. Cultures were plated in
triplicate at 75 to 120 × 103 MNC/mL. In some
experiments a neutralizing antibody against IL-10 was preincubated with
IL-10 for 2 hours at room temperature. Neutralizing antibodies against
GM-CSF, G-CSF, IL-3, SCF, or IGF-1 were used as recommended by the
manufacturer. Plates were incubated at 37°C, 5% CO2,
and full humidity. After a culture period of 14 days, cultures were
examined under an inverted microscope. Aggregates with at least 50 hemoglobinized cells, easily recognizable by their red color, were
counted as BFU-E-derived erythroid bursts. For cultivation of BFU-E
from normal individuals, 2 U/mL EPO was added to culture dishes.
Semiquantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) analysis of GM-CSF transcripts.
A total of 2 × 107 PV PBMNCs were cultured in
suspension both with and without IL-10 (10 ng/mL) for 48 hours. After
incubation, cells were washed twice in diethylpyrocarbonate-treated
water and 107 cell aliquots were lysed by addition of 1.6 mL RNAzol B (Biotecx, Houston, TX). Total RNA was extracted as
described.18 The integrity of RNA was controlled by
electrophoresis through formaldehyde agarose gels. High quality RNA was
quantitated by measuring absorbance at 260 nm and 1 g total RNA was
subjected to cDNA synthesis as recently described.19
For semiquantitative analysis of GM-CSF mRNA, an RT-PCR technique that
allows measurements of relative transcript levels was applied.20,21 The oligonucleotide primer sequences for
amplification of GM-CSF were 5 -CTGCTGCTGAGATGAATGAAACAG-3 and
5-TGGACTGGCTCCCAGCAGTCAAAG-3, which bracketed a GM-CSF fragment of
286 bp.22 PCR amplification of ABL (Abelson)
transcripts was used as a reference to assess variation of total RNA or
cDNA between samples. The primer sequences for amplification of ABL
were as follows: 5-CAGCGGCCAGTAGCATCTGACTTTG-3 and
5-CCATTTTTGGTTTGGGCATCACACCATTCC-3 resulting in the production of a
PCR fragment of 228 bp.19 The linear ranges of PCR
amplifications of GM-CSF and ABL were established as a function of
cycle number and cDNA concentration as described.15,20,21
Reaction conditions included 3 L cDNA, 20 pmol of each primer, 1.5 mmol/L MgCL2, 200 mol/L of each dNTP, 2.5 U Ampli Taq DNA Polymerase
(Perkin Elmer Cetus, Norwalk, CT), and (32P) dCTP (150,000 cpm) in a 50-L reaction volume. The thermal cycling conditions were denaturation at 94°C (1 minute), annealing at 60°C (1 minute), and extension at 72°C (2 minutes), preceded by an initial denaturation step at 94°C for 5 minutes, and followed by
a terminal extension of 10 minutes at 72°C. The number of PCR cycles for amplification of GM-CSF and ABL transcripts was 32 and 25 cycles, respectively. Reaction products were subjected to 6%
polyacrylamide gels (Novex, San Diego, CA), and dried gels were exposed
to Kodak XAR-5 films (Eastman Kodak, Rochester, NY) at
 70°C for 12 hours.
For quantification of PCR products incorporated (32P) dCTP
was measured on autoradiograms by using the Bio Rad 670 Imaging
Densitometer and the system`s volume integration program (Bio Rad Gel
DOC 1000 system, Molecular Analyst/PC software; BioRad,
Richmond, CA). Potential differences of total cellular RNA/cDNA in PCR
analyses were corrected by dividing GM-CSF values by the ABL value
(mean value of six PCR analyses). The relative level of GM-CSF
transcripts was measured in patient samples in three PCR analyses in
duplicate using freshly synthesized cDNA.
Statistical analysis.
The t-test was used to determine the significance of
differences. A P value of <.05 was considered statistically
significant.
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RESULTS |
Inhibitory effect of IL-10 on autonomous BFU-E growth in patients with
PV.
As it has been originally shown by Prchal and Axelrad,1
erythroid colonies can grow in methylcellulose cultures containing PBMNCs from patients with PV in the absence of exogenous EPO. Treatment
of PV cell cultures with IL-10 resulted in a profound and
dose-dependent inhibition of autonomous BFU-E growth
(Fig 1). The inhibitory effect of IL-10
became apparent at a concentration of 1 ng/mL and was even more
pronounced at higher concentrations. This effect was specific, because
a neutralizing anti-IL-10 antibody was able to prevent IL-10-induced
suppression of BFU-E growth (Fig 1). The effect of 10 ng/mL IL-10 on
autonomous BFU-E growth was investigated in PBMNCs from 5 patients with
PV (Table 2). As shown in Table 2,
autonomous formation of erythroid colonies greatly varied among
different patients but was significantly inhibited by IL-10 in all of
them. On average, 10 ng/mL of IL-10-inhibited EPO independent BFU-E
growth from PV PBMNCs by 81% (range, 71% to 94%).
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| Fig 1.
Dose-dependent inhibitory effect of IL-10 on autonomous
BFU-E growth from PV cells and its abrogation by an anti-IL-10 antibody
in patient HR. A total of 120 × 103 PBMNC/mL were
cultured in methylcellulose with medium alone, with increasing
concentrations of IL-10, or with IL-10 plus a neutralizing antibody
against IL-10. Colony growth was assessed after 14 days. Results
represent the mean values ± standard deviation (SD) from triplicates.
* Significant change from control with P value at least
<.05.
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Effect of anticytokine antibodies on autonomous BFU-E growth from PV
cells.
To elucidate the possible mechanism of the inhibitory action of IL-10,
we first tried to identify the factor responsible for autonomous BFU-E
growth in PV by adding neutralizing antibodies against GM-CSF, IL-3,
G-CSF, SCF, and IGF-1 to cell cultures from three patients with marked
colony growth (Fig 2). Not unexpectedly the
IGF-1 antibody significantly inhibited autonomous BFU-E growth in our
FCS containing culture system (patient SA), because IGF-1 has been
reported as the major EPO-like activity in FCS.23 Among the
other anticytokine antibodies tested only the anti-GM-CSF antibody
significantly decreased spontaneous growth of erythroid colonies in all
three patients tested, with a mean inhibition by 52% (range, 42% to
63%). This clearly suggested that GM-CSF and IGF-1 were involved in
the spontaneous formation of erythroid colonies from PV PBMNCs in this
culture system.
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| Fig 2.
Effect of anticytokine antibodies on autonomous BFU-E
growth from PV cells in three patients. PBMNCs were cultured in
methylcellulose with medium alone or with antibodies against GM-CSF,
IL-3, G-CSF, SCF, or IGF-1, respectively. Colony growth was assessed
after 14 days. Results represent mean values ± SD from triplicates. *
Significant change from control with P value at least <.05.
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Effect of exogenous growth factors on IL-10-induced suppression of
autonomous BFU-E growth from PV cells.
The fact that IL-10 has been shown to inhibit cytokine synthesis in
different cell types of the mononuclear cell fraction4,13 led us to hypothesize that inhibition of autonomous BFU-E growth by
IL-10 was secondary to IL-10-induced suppression of endogenous release
of growth regulatory molecules. If this was the case one would expect
that exogenous addition of particular cytokines could at least in part
reverse growth inhibition by IL-10. In contrast, restoration of colony
growth by exogenous growth factors would not be observed if IL-10 had a
direct cytotoxic effect on PV cells. In fact, exogenous GM-CSF and IL-3
significantly restimulated erythroid colony formation in IL-10 treated
cultures (Fig 3). All other cytokines were
not effective in correcting IL-10-induced growth inhibition. Also IGF-1
alone at concentrations up to 100 ng/mL was not able to restore
autonomous BFU-E growth in IL-10 suppressed cultures, nor did it
significantly potentiate the effects of GM-CSF when added in
combination (Table 3). This observation and
the suppressive effect of the anti-IGF-1 antibody in the culture system
suggest that IGF-1 is important for EPO-independent erythroid colony
formation in PV patients but is not involved in growth inhibition by
IL-10.
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| Fig 3.
Effect of exogenous growth factors on IL-10-induced
suppression of BFU-E growth from PV cells. PBMNCs were cultured in
methylcellulose with 10 ng/mL IL-10 in the presence or absence of
exogenous GM-CSF (100 U/mL), IL-3 (10 U/mL), G-CSF (100 U/mL), SCF (10 ng/mL), and IGF-1 (10 ng/mL), respectively. Colony growth was assessed
after 14 days. Results represent mean values ± SD from triplicates. *
Significant change from IL-10 treated cultures with P value at
least <.05.
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Table 3.
Effect of IGF-1 Alone or in Combination With GM-CSF
on IL-10-Induced Suppression of Autonomous BFU-E Growth From PV
Cells
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Inhibitory effect of IL-10 on GM-CSF expression in PV PBMNCs.
The demonstration of both the antiproliferative action of the
anti-GM-CSF antibody on the autonomous BFU-E growth and the restoration
of IL-10-induced growth suppression by exogenous GM-CSF strongly
suggested that the growth inhibitory effect of IL- 10 in PV was at
least in part caused by suppression of spontaneous GM-CSF production.
Therefore, the effect of IL-10 on GM-CSF expression in unseparated
PBMNCs from a patient with PV (HR) was studied by a semiquantitative
PCR technique. We have previously shown that the PCR technique used by
us allows at least semiquantitative measurements of GM-CSF transcript
levels by establishing linear ranges of amplifications of GM-CSF as a
function of cycle number and cDNA concentrations.15 After
culturing PV cells in the presence of IL-10 (10 ng/mL) for 48 hours,
GM-CSF transcript levels were substantially lower than that of cells
kept in suspension without IL-10 (Fig 4A).
In comparison, ABL transcripts, which served as a control, remained
unchanged during the time of culture (Fig 4B). Figure 4C shows
corrected GM-CSF mRNA levels in PV PBMNCs at 48 hours. Comparison of
corrected GM-CSF transcript levels between PBMNCs cultured with and
PBMNCs cultured without IL-10 showed a mean decrease by 47%.
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| Fig 4.
Semiquantitative RT-PCR analysis of GM-CSF transcript
levels. (A) Autoradiogram showing incorporated radioactivity of
amplification products obtained from PV PBMNCs cultured in suspension
with or without IL-10 for 48 hours. (B) Autoradiogram showing ABL
transcripts that served as a reference to correct for potential
variations of RNA or cDNA samples. (C) Corrected GM-CSF transcript
levels in cultured PV cells. Each sample was analyzed in three
radioactive PCR analyses in duplicate using freshly synthesized cDNA.
The quantity of 32P incorporated into PCR product was
determined by densitometric scanning of the autoradiograms. Results
were corrected by dividing GM-CSF values by the mean values obtained
from six ABL transcripts of that cDNA. The results are shown as mean
values.
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Effect of IL-10 on EPO-dependent BFU-E growth in normal individuals.
To investigate the effect of IL-10 on the EPO-dependent in vitro growth
of normal erythroid progenitors, PBMNCs from five healthy volunteers
were cultured in the presence of EPO with or without 10 ng/mL IL-10
(Table 4). IL-10-induced suppression of EPO-dependent erythroid colony growth was seen in all five individuals, but this inhibition was less pronounced than in PV patients. The mean
inhibition of normal EPO-dependent BFU-E growth by 10 ng/mL IL- 10 was
45% ± 12% (standard deviation), which was significantly less than
the inhibition of EPO-independent erythroid colony growth in PV
patients ( 81% ± 12%, P < .001)
| |
DISCUSSION |
Elegant studies by Adamson et al24 analyzing the isoenzyme
pattern in different cell types of PV patients heterozygous for G6PD
have shown that circulating red blood cells, granulocytes, and
platelets expressed the same isoenzyme, suggesting that these cells
were unicellullar in origin and derived from an abnormal multipotential
progenitor cell. Later, Prchal et al25 could show that
erythroid colonies from PV patients that formed in the absence of
exogenous EPO contained the same G6PD isoenzyme type as that expressed
by peripheral blood elements. Thus, the so-called endogenous colonies
arose from the abnormal clone that was responsible for the
overproduction of red blood cells in PV. When exogenous EPO was added,
increasing numbers of erythroid colonies were formed containing cells
that expressed the other G6PD isoenzymes, indicating the existence of
both malignant and nonmalignant populations of hematopoietic progenitor
cells in PV marrow. Here we show a profound inhibitory effect of IL-10
on endogenous erythroid colony formation in vitro in patients with PV.
A significant inhibition of spontaneous erythroid colony formation by
IL-10 was observed in all five PV patients investigated. The effect of
IL-10 was dose-dependent and specific, because a neutralizing
anti-IL-10 antibody was able to prevent IL-10-induced suppression of
BFU-E growth.
The mechanism of spontaneous erythroid colony formation in vitro and
the factors that lead to in vivo expansion of clonal cells in patients
with PV remain to be fully understood. Spontaneous erythroid colony
formation in the presence of anti-EPO antibodies (3) and in serum-free
culture conditions (2) exclude minute amounts of EPO in the culture
system as a potential stimulus for endogenous colony formation. The
marked reduction of spontaneous erythroid colony formation by cells
from PV patients by depletion of monocytes and its restoration by
readdition of adherent cells or adherent cell
supernatant26,27 strongly suggests monocyte-derived
molecules as stimulatory factors for erythroid colony formation in PV.
Several cytokines including GM-CSF,28 IL-3,28
SCF,29 and IGF-130 have been shown to enhance
EPO independent BFU-E growth if added to semisolid cultures containing
PV MNCs. Moreover a hypersensitivity of PV BFU-E to each of these
growth factors compared with normal erythroid progenitors has been
shown,28-30 which may be a principal factor in the profound
marrow hyperplasia and increased blood counts of patients with PV.
Recently, a defect in phosphatase activity in PV cells has been
proposed as the molecular basis for the enhanced response of PV BFU-E
to growth regulatory molecules. Thus, increased basal and induced
tyrosine phosphorylation of the IGF-1 receptor  subunit in
circulating MNCs in such patients has been reported.31
Moreover, a diminished enhancement of erythroid colony formation in the
presence of orthovanadate,32 an inhibitor of protein
tyrosine phosphatases, in PV cells has been shown.
There is substantial evidence that the inhibitory effect of IL-10 on
autonomous BFU-E growth in PV is indirect. Spontaneous formation of
erythroid colonies from PV cells has been shown to be decreased after
removal of adherent cells.26,27 In unseparated PV PBMNCs,
we found, in agreement with observation by others,33 that
autonomous BFU-E growth was markedly reduced by addition of an
anti-GM-CSF antibody, suggesting spontaneous release of GM-CSF as an
important mechanism of EPO-independent erythroid colony formation. Not
unexpectedly, an anti-IGF-1 antibody also inhibited autonomous BFU-E
growth in our culture system because IGF- 1 has been shown to be the
major EPO-like activity in FCS.23 The antiproliferative
action of anti-GM-CSF antibody on EPO-independent BFU-E formation in PV
and the fact that IL-10 has been shown to inhibit cytokine synthesis,
including GM-CSF in human monocytes,13 led us to
hypothesize that inhibition of BFU-E growth by IL-10 was mainly caused
by IL-10-induced suppression of endogenous GM-CSF release. Our
observation that exogenous GM-CSF was able to, at least in part,
reverse growth inhibition by IL-10 strongly supports this concept. In
fact, analysis of the effect of IL-10 on GM-CSF expression in MNCs from
a patient with PV clearly showed that the IL-10-induced inhibition of
erythroid colony growth correlated with a substantial decrease in
GM-CSF transcripts in PV PBMNCs. Exogenous IL-3 also restimulated
autonomous BFU-E formation in IL-10-treated PV cell cultures, but an
anti-IL-3 antibody had no antiproliferative effect on spontaneous
growth of erythroid progenitors. These data suggest that in addition to
GM-CSF-responsive PV BFU-E, there seems to exist an IL-3-responsive
population, which however, cannot be sufficiently stimulated by the
endogenously released growth factors in unstimulated cultures.
The inhibitory effect of IL-10 on hematopoietic colony formation is not
specific for PV BFU-E. However the growth suppression of IL-10 on
EPO-independent erythroid colony growth in PV seems to be more
pronounced than that on EPO-dependent normal BFU-E. In contrast to a
mean growth inhibition by 81% on autonomous BFU-E formation from PV
PBMNCs, IL-10 decreased the number of EPO-stimulated BFU-E by
approximately 45% in PBMNC cultures from normal individuals. Moreover,
the inhibitory action of IL-10 is not restricted to the erythroid
lineage, because we have also shown IL-10-induced inhibition of
spontaneous formation of myeloid colonies in normal individuals15 and patients with CMML16 through
suppression of endogenous GM-CSF release. Thus, the inhibition of
GM-CSF synthesis by IL-10 makes it useful in indirectly modulating
hematopoiesis in a variety of conditions.
Despite its well-defined effects in vitro, the in vivo role of GM-CSF
in normal and abnormal hematopoiesis is much less clear. Mice deficient
in GM-CSF (GM-/-) through homologous recombination in
embryonal stem cells show no alterations in hematopoiesis34
suggesting substantial overlap of function with other
colony-stimulating factors. On the other hand, animals transplanted
with cells containing the GM-CSF cDNA developed a fatal syndrome
resembling myeloproliferation, which however, was non-neoplastic in
nature.35 In PV patients it is not known if GM-CSF, as in
the in vitro system, plays a significant role in the expansion of
clonal cells. If this is the case, therapy with IL-10, a cytokine that
has been well tolerated in first clinical trials,36,37
could be considered as a strategy to suppress the excessive production
of red blood cells in PV.
| |
FOOTNOTES |
Submitted September 22, 1997;
accepted April 20, 1998.
Supported by the "Medizinisch-Wissenschaftlicher Fonds des
Bürgermeisters der Bundeshauptstadt Wien."
Address reprint requests to Klaus Geissler, MD, 1st Medical Department,
Division of Hematology, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract
