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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1506-1508
BRIEF REPORT
The Exodus subfamily of CC chemokines inhibits the
proliferation of chronic myelogenous leukemia progenitors
Robert Hromas,
Larry Cripe,
Giao Hangoc,
Scott Cooper, and
Hal E. Broxmeyer
From Hematology/Oncology, Biochemistry/Molecular Biology,
Microbiology/Immunology, and the Walther Oncology Center, Indiana
University Medical Center, Indianapolis, Indiana.
 |
Abstract |
Chemokines are a family of related proteins that regulate leukocyte
infiltration into inflamed tissue and play important roles in disease
processes. Among the biologic activities of chemokines is inhibition of
proliferation of normal hematopoietic progenitors. However, chemokines
that inhibit normal progenitors rarely inhibit proliferation of
hematopoietic progenitors from patients with chronic myelogenous
leukemia (CML). We and others recently cloned a subfamily of CC
chemokines that share similar amino-terminal peptide sequences and a
remarkable ability to chemoattract T cells. These chemokines,
Exodus-1/LARC/MIP-3 , Exodus-2/SLC/6Ckine/TCA4, and
Exodus-3/CK 11/MIP-3, were found to inhibit proliferation of
normal human marrow progenitors. The study described here found that
these chemokines also inhibited the proliferation of progenitors in
every sample of marrow from patients with CML that was tested. This
demonstration of consistent inhibition of CML progenitor proliferation
makes the 3 Exodus chemokines unique among chemokines.
(Blood. 2000;95:1506-1508)
© 2000 by The American Society of Hematology.
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Introduction |
Chemokines are a family of structurally related
proteins that are the major mediators of all leukocyte
migration.1-4 The chemokine family is subdivided into 2 major groups on the basis of how the first 2 conserved cysteines are
arranged. If the first 2 cysteines are separated by a single amino
acid, the chemokines are considered to be in the CXC family
(alternatively called  ). If the first 2 cysteines are immediately
adjacent to each other, the chemokines are classified in the CC family
(alternatively called ).
Many studies have found that chemokine receptors are important in human
disease.1-4 For example, HIV coreceptors (with CD4) were
identified as chemokine receptors.5-7 Because of the
ability of chemokines to stimulate leukocyte infiltration, they play
crucial roles in many diseases in which there is inflammatory tissue
destruction, such as adult respiratory distress syndrome, myocardial
infarction, rheumatoid arthritis, and atherosclerosis.8-11
CXC chemokines that bind to and activate the chemokine receptor CXCR2
can stimulate angiogenesis, whereas CXC chemokines that bind to CXCR3
inhibit angiogenesis.1-4
We and others have shown that many but not all chemokines from both the
CC and CXC family can negatively regulate normal hematopoietic progenitor proliferation.12-18 This inhibition occurs with
both human and murine normal marrow progenitors and both in vitro and in vivo. However, we and other investigators have found that this inhibition of progenitor proliferation does not always extend to
chronic myelogenous leukemia (CML).13,16-18
In this study, we demonstrated that progenitor proliferation in marrow
from patients with CML can be significantly and consistently inhibited
by a subfamily of CC chemokines that we and others have isolated and
that suppress proliferation of normal progenitors.13 This
subfamily of CC chemokines are termed
Exodus-1/LARC/MIP-3,14 Exodus-2/SLC/6Ckine/TCA4,15
and Exodus-3/ELC/CK11/MIP-3 (see Yoshie et al19
and Zlotnik et al20 for a review of all 3).
| |
Study design |
Patient samples
Institutional review board-approved informed consent was obtained
from patients with CML. All patients were in the chronic phase, and
none were taking interferon- (IFN-). Marrow was aspirated with
the patient under local anesthesia and was placed in heparin-coated tubes. All patients had cytogenetic analysis to confirm the diagnosis of CML. In all patients, 100% of metaphases analyzed were positive for
t(9;22).
Hematopoietic progenitor assays
Hematopoietic colony-formation assays were performed essentially as
previously described.13-15 Low-density mononuclear cells were obtained from heparinized patient marrow by means of Ficoll gradient centrifugation. Low-density human marrow mononuclear cells at
a concentration of 5 × 104/mL were plated in 1%
methylcellulose in Iscove's modified Dulbecco's medium supplemented
with 30% fetal-calf serum, pure recombinant human erythropoietin (1 U/mL), pure recombinant IL-3 (100 U/mL), and pure recombinant Steel
factor (50 ng/mL) for analysis of colony-forming units
granulocyte-macrophage (CFU-GM), colony-forming units
granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM), and
burst-forming units erythrocyte (BFU-E). Purified recombinant Exodus
chemokine proteins (R&D, Minneapolis, MN) were compared with purified
recombinant macrophage inflammatory protein-1 (MIP-1), another CC
chemokine known to inhibit normal marrow progenitor proliferation but
which has shown little activity against CML progenitor proliferation.
The concentration chosen after extensive preliminary studies was 100 ng/mL.13-18 This concentration elicits maximal inhibition
of normal progenitor-cell proliferation. The percentage of progenitors
in S phase at the time of the chemokine treatment was analyzed by
tritiated thymidine kill assays as previously described.13-18 Tritiated thymidine is preferentially taken
up by cells in S phase and is cytotoxic to those cells because of its
radioactivity. Chemokine inhibitory activity is only effective against
progenitors in S phase of the cell cycle.12,17
| |
Results and discussion |
Marrow from 2 patients with CML in the chronic phase who had not
received treatment with IFN was tested for inhibition of hematopoietic progenitor proliferation by using various concentrations of Exodus-1, Exodus-2, and Exodus-3 to establish a dose-response curve
(Figure 1). MIP-1 at concentrations
ranging from 1.25 ng/mL to 500 ng/mL did not
have any inhibitory effect on the proliferation of
progenitors in the samples from these patients. However, all 3 Exodus
chemokines produced a dose-dependent inhibition of CFU-GM, BFU-E, and
CFU-GEMM progenitor proliferation in both patient samples, beginning at
a concentration of 12.5 ng/mL (Figure 1). At 1.25 ng/mL, there was
little inhibition. The dose-response curve was similar for all the
Exodus chemokines. When both CML patient samples were taken
into account, this inhibitory response leveled off at between 50 ng/mL and 100 ng/mL. Therefore, for more extensive studies, we chose
100 ng/mL as the concentration of Exodus-1, Exodus-2, and Exodus-3
chemokine to obtain maximal response in all progenitor assays.
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| Fig 1.
Dose-response curves of the inhibition of proliferation
of CFU-GM, BFU-E, and CFU-GEMM by Exodus-1, Exodus-2, and Exodus-3.
Progenitor assays of marrow from 2 newly diagnosed, untreated patients
with chronic myelogenous leukemia (CML) were performed with varying
concentrations of the Exodus chemokines. MIP-1 served as a negative
control and did not inhibit CFU-GM proliferation to any appreciable
extent, even at concentrations of up to 500 ng/mL. Data are
presented as the average of the percentage of
proliferative inhibition from triplicate cultures compared with control
average colony formation without chemokine treatment. Mean (± SD) control colonies were 22 ± 3 for CFU-GM, 126 ± 14 for
BFU-E, and 13 ± 3 for CFU-GEMM for CML patient 1 and
195 ± 10 for CFU-GM, 168 ± 10 for BFU-E, and 49 ± 5
for CFU-GEMM for CML patient 2.
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Marrow was obtained from 13 additional patients with CML in the chronic
phase who were not taking IFN-. Tritiated thymidine kill assays were
used to test the marrow samples for the percentage of progenitors in S
phase. In these samples, an average of 56% of CFU-GM, 49% of BFU-E,
and 53% of CFU-GEMM progenitor cells were in S phase (Figure
2).
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| Fig 2.
Influence of Exodus subfamily of CC chemokines on colony
formation of progenitors in marrow from patients with CML.
Data are presented as the percentage of inhibition compared with
control colony formation (without chemokine treatment) for each
patient. Mean (± SD) control colonies were 69 ± 83 for
CFU-GM, 85 ± 55 for BFU-E, and 35 ± 36 for
CFU-GEMM. For the CML patient samples, data are presented
as average values, with SD bars. If no SD bars are shown, the SD was
too small to be included on the figure. For Exodus-1, n = 13; for
Exodus-2, n = 4; for Exodus-3, n = 3; and for MIP-1, n = 11.
Two CML patient samples in which MIP-1 inhibited the progenitors are
not on the figure but are discussed in the text.
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Next, the effect of MIP-1 on CML progenitor-cell proliferation was
tested. MIP-1 inhibits normal marrow progenitor-cell proliferation
but most often does not act on CML progenitors.16-18 As
expected, MIP-1 did not significantly inhibit progenitor
proliferation in 11 of the 13 CML marrow samples (Figure 2). However,
in 2 samples, MIP-1 did inhibit progenitor proliferation: the
average percentage of inhibition in these samples compared with
untreated controls was 43% for CFU-GM, 37% for BFU-E, and 50% for
CFU-GEMM. In contrast, in all 13 CML samples, progenitor proliferation
was significantly inhibited by Exodus-1 (P < .01). CFU-GM
were inhibited by an average of 53%, BFU-E by an average of 47%, and
CFU-GEMM by an average of 52% (Figure 2).
Because Exodus-2 and Exodus-3 were isolated and characterized some time
after Exodus-1, fewer CML samples were analyzed with those chemokines.
In the 4 CML marrow samples tested, Exodus-2 significantly inhibited
(P < .01) CFU-GM proliferation by an average of 61%, BFU-E
by an average of 49%, and CFU-GEMM by an average of 50%. In the 3 CML
samples tested, Exodus-3 significantly inhibited (P < .01)
CFU-GM proliferation by an average of 57%, BFU-E by an average of
50%, and CFU-GEMM by an average of 48%.
Thus, progenitor proliferation in all the samples of marrow from
patients with CML we tested was significantly inhibited by all 3 Exodus subfamily chemokines. There were no significant
differences between the 3 chemokines in their ability to maximally
inhibit progenitor proliferation. All 3 Exodus chemokines were markedly more effective than MIP-1 at inhibiting CML progenitor
proliferation. Exodus-1 uses CCR6 as its receptor and Exodus-2 and
Exodus-3 use CCR7. The data presented here offer the possibility that
activation of CCR6 and CCR7 may inhibit proliferative signals in
hematopoietic progenitors in patients with CML.21
Much of the morbidity of CML in the chronic phase is
due to the pancytosis that results from a proliferative advantage
that CML progenitors have over normal marrow progenitors. Use
of the Exodus chemokines may be an effective approach to
controlling blood-cell production in CML. The possibility that the
Exodus chemokines could produce cytogenetic remissions awaits a
clinical trial. It would be especially interesting to see whether the
Exodus chemokines can produce responses in chronic-phase patients
with CML in whom treatment with IFN- has failed.
| |
Footnotes |
Submitted April 21, 1999; accepted October 12, 1999.
Supported by US Public Health Service grants RO1 HL56416 and DK53674 to
H.E.B. and RO1 HL48914 to R.H., who is also supported by a Leukemia
Society of America Scholar Award and a Translational Research Award.
Reprints: Robert Hromas, Walther Oncology Center, Indiana
University Medical Center, R4-202, 1044 W Walnut St, Indianapolis, IN
46202; e-mail: rhromas{at}iupui.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction