By
From the * Department of Microbiology/Immunology, the Department of Medicine, and the Walther
Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202; the Walther
Cancer Institute, Indianapolis, Indiana 46208; and the § Laboratory of Host Defenses, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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Macrophage inflammatory protein (MIP)-1, a CC chemokine, enhances proliferation of mature subsets of myeloid progenitor cells (MPCs), suppresses proliferation of immature MPCs,
and mobilizes mature and immature MPCs to the blood. MIP-1
binds at least three chemokine receptors. To determine if CCR1 was dominantly mediating the above activities of MIP-1
,
CCR1-deficient (
/
) mice, produced by targeted gene disruption, were used. MIP-1
enhanced colony formation of marrow granulocyte/macrophage colony-forming units (CFU-GM),
responsive to stimulation by granulocyte/macrophage colony-stimulating factor (GM-CSF),
and CFU-M, responsive to stimulation by M-CSF, from littermate control CCR1+/+ but not
CCR1
/
mice. Moreover, MIP-1
did not mobilize MPCs to the blood or synergize with
G-CSF in this effect in CCR1
/
mice. However, CCR1
/
mice were increased in sensitivity to MPC mobilizing effects of G-CSF. Multi-growth factor-stimulated MPCs in CCR1
/
and CCR1+/+ marrow were equally sensitive to inhibition by MIP-1
. These results implicate
CCR1 as a dominant receptor for MIP-1
enhancement of proliferation of lineage-committed
MPCs and for mobilization of MPCs to the blood. CCR1 is not a dominant receptor for
MIP-1
suppression of MPC proliferation, but it does negatively impact G-CSF-induced
MPC mobilization.
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Introduction |
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Mature blood cells must be constantly replaced. These cells are replenished from hematopoietic stem and progenitor cells (1). Intrinsic to this process is an interacting network of cytokines that control production and movement of stem and progenitor cells. Identifying the cytokines and specific receptors involved in these processes is important for clinically modulating blood cell production and movement.
Macrophage inflammatory protein (MIP)-1, a cysteine
cysteine (CC) chemokine (2), enhances and inhibits proliferation of myeloid progenitor cells (MPCs) and mobilizes
MPCs to the blood. MIP-1
was first shown to enhance
colony formation of granulocyte/macrophage (CFU-GM)
and macrophage (CFU-M) progenitor cells in vitro (6, 7), a
direct effect on mature subsets of MPCs that responded to
stimulation by GM-CSF or M-CSF. By itself, MIP-1
did
not stimulate colony formation by any type of MPC. The
enhancing effect of MIP-1
on lineage-committed cells
was subsequently confirmed by others (8). MIP-1
was
subsequently shown to suppress more immature subsets of
cells, including CFU-spleen (CFU-S [11], considered a stem,
but not long-term marrow repopulating, cell) and immature MPCs (CFU-GM, erythroid [BFU-E], and multipotential [CFU-GEMM] progenitors) that proliferated in response to erythropoietin (Epo), IL-3, GM-CSF, and steel
factor (SLF) (7, 12, 13). The suppressive effects were
direct on MPCs (7, 12, 13). MIP-1
suppression was substantiated in vivo in mice (8, 14) and was confirmed in
patients with breast cancer undergoing a phase I clinical
trial with BB10010 (15), an MIP-1
analogue. BB10010
was also a modest mobilizing agent for stem and progenitor
cells into the blood of mice (18); BB10010 synergized in
this effect with G-CSF (18), a stem and progenitor cell mobilizer used clinically (1). The MPC mobilizing capacity of
BB10010 was confirmed in a human clinical trial (15).
Chemokines act through seven transmembrane G protein-linked receptors (2). 16 chemokine receptors have
been identified that bind known chemokines, including 9 in the CC chemokine group (2). Since MIP-1 binds
chemokine receptors CCR1, CCR5, and D6, it is not
clear through which chemokine receptor(s) MIP-1
activities are mediated. Chemokines do not appear to signal
through D6 (19), suggesting that CCR1, CCR5, and/or
an unknown receptor for MIP-1
may be involved in the
above-noted activities of MIP-1
on MPCs. CCR1
/
mice have been developed by targeted gene disruption (20), and have demonstrated certain nonredundant functions in
hematopoiesis, host defense, and inflammation. These mice
were used in this study to demonstrate that CCR1 is a
dominant receptor for enhancement of proliferation of mature MPCs and mobilization of MPCs to blood, but not for
suppression of proliferation of immature MPCs.
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Materials and Methods |
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Mice.
Generation of CCR1Cytokines.
Purified recombinant preparations of cytokines were used. Human (hu) and murine (mu) MIP-1MPC Assays.
Colony assays were done as described elsewhere (22). Unseparated bone marrow (5 × 104 cells/ml) and low-density blood cells (1-2 × 105 cells/ml, obtained after density cut procedure) were isolated from mice (22). To assess whether MIP-1In Vivo MPC Mobilization Assay.
Mice were given either control diluent, huG-CSF, huMIP-1Statistical Analysis.
Results are given as mean ± SEM, and Student's t test was used to analyze the data. P values < 0.05 designated significant differences between test points. ![]() |
Results |
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To determine if CCR1 was a dominant receptor for MIP-1 enhancement of colony formation (6, 7), we tested mu and
hu forms of MIP-1
. As shown in Fig. 1, mu and huMIP-1
significantly enhanced colony formation by CCR1+/+,
but not by CCR1
/
, marrow cells stimulated to proliferate by either GM-CSF or M-CSF. Colonies formed in the
presence of GM-CSF, with or without MIP-1
, were
composed mainly of granulocytes and macrophages with
<20% of the colonies containing only granulocytes or macrophages. No shifts in colony types were noted in the
absence or presence of MIP-1
. Colonies formed with
M-CSF, with or without MIP-1
, were composed of macrophages. No colonies formed in the absence of GM-CSF
or M-CSF whether or not MIP-1
was added to the plates. This suggests that CCR1 acts as a dominant receptor for
the MIP-1
enhancing effects on MPCs stimulated by
GM-CSF or M-CSF.
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MIP-1 suppresses MPCs stimulated to proliferate by
combinations of growth factors (7, 12, 13). One report
using antibodies to CCR1 suggested that the suppressing
effects of MIP-1
on colony formation by BFU-E were
mediated by CCR1 (23). To determine if CCR1 was a
dominant receptor for MIP-1
suppression, we analyzed the effects of MIP-1
on colony formation by marrow cells
from CCR1+/+ and CCR1
/
mice stimulated to proliferate with Epo, PWMSCM, SLF, and hemin. As shown
in Fig. 2, both mu and huMIP-1
were equally potent in suppressing colony formation by CFU-GM, BFU-E, and
CFU-GEMM from CCR1+/+ and CCR1
/
marrow cells.
As controls, other CC (MCP-1 and Exodus-1) and CXC
(IL-8, muMIP-2, ENA-78, IP-10, and PF4) chemokines
known to be inhibitory under these conditions (5) were
tested and found to be equally suppressive on CCR1+/+
and CCR1
/
MPCs (Fig. 2). Chemokines known to be
nonsuppressive (5; GRO-
, GRO-
, NAP-2, RANTES,
and MIP-1
) did not inhibit colony formation of CCR1+/+
or CCR1
/
MPCs (Fig. 2). These results suggest that CCR1
is not a dominant receptor for MIP-1
suppression of
multi-growth factor-stimulated MPCs.
|
G-CSF and BB10010, an
MIP-1 analogue, induce mobilization of stem and MPCs
to blood (1, 18) although with different kinetics, and G-CSF
and BB10010 in combination are additive or synergize in
this mobilization (18). In preliminary experiments using
C3H/Hej mice, we confirmed that huMIP-1
induction
of mobilization of MPCs to blood was rapid (within 15 min
to 1 h) and reversible. huRANTES did not have a mobilizing effect on MPCs in these same experiments. We thus assessed the in vivo MPC mobilizing effects of MIP-1
and
G-CSF. As shown in Fig. 3, MIP-1
and G-CSF each significantly mobilized MPCs to the blood of CCR1+/+ mice,
and the combination of G-CSF with MIP-1
showed
greater mobilization than either cytokine alone. In contrast,
MIP-1
did not significantly enhance mobilization of
MPCs to the blood of CCR1
/
mice (Fig. 3). Moreover,
MIP-1
did not act with G-CSF to enhance mobilization
further in CCR1
/
mice. However, MPCs in CCR1
/
mice were more sensitive to the mobilizing effects of G-CSF
alone than in CCR1+/+ mice. In 3 separate experiments in
which 4-5 mice per group per experiment were assessed,
1.3-, 1.7-, and 11.6-fold more CFU-GM, 1.5-, 2.3-, and
4.9-fold more BFU-E, and 2.6- and 3.4-fold more CFU-GEMM were mobilized in CCR1
/
compared with
CCR1+/+ mice. In one experiment, we did not detect
greater mobilization of CFU-GEMM in CCR1
/
mice.
These results suggest that CCR1 is a dominant receptor for
the MPC mobilizing effects of MIP-1
; moreover, CCR1
appears to play a negative role in G-CSF-induced mobilization of MPCs to the blood.
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Discussion |
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Since several chemokine receptors can bind more than
one chemokine and some chemokines can bind more than
one chemokine receptor (2), it is not always clear which
chemokine receptor mediates the effects of specific chemokines. MIP-1 binds to three chemokine receptors: CCR1,
CCR5, and D6. Although D6 does not elicit a Ca2+ influx
signal in response to MIP-1
or other chemokines that bind
this receptor (19), it is possible that other intracellular signals
are activated through D6 in response to certain chemokines, and there is the possibility that additional receptors will
be identified that bind MIP-1
. The availability of CCR1
/
mice (20) allowed us to assess if CCR1 served as a dominant receptor for three previously reported functions of
MIP-1
. The results presented here clearly demonstrate that
in cells without functional CCR1, MIP-1
did not enhance
proliferation of CFU-GM stimulated by GM-CSF, or
CFU-M stimulated by M-CSF, nor did MIP-1
induce in
vivo mobilization of MPCs to the blood, implicating CCR1
as a dominant receptor for these activities. The MIP-1
- induced MPC mobilization effects complement our previous studies in which CCR1 was shown to be a dominant
receptor for bacterial LPS-induced movement of MPCs
between bone marrow, spleen, and blood and for MIP-1
mobilization of neutrophils to the blood (20).
Our current studies with MIP-1 confirm the mobilization effects on MPCs noted by others using an MIP-1
analogue, BB10010 (18), as well as the additive/greater than
additive mobilization apparent when BB10010 is given as a
single injection to mice previously injected with G-CSF.
G-CSF mobilizes stem and progenitor cells for autologous
and allogeneic transplantation (1), and enhancement of this
may be clinically important. We also found that CCR1
/
mice were more sensitive to the MPC mobilizing effects of
G-CSF than were CCR1+/+ mice. This unexpected finding
implicates CCR1 as a negative component in G-CSF-
induced MPC mobilization. How CCR1 would negatively mediate such an effect is not clear. It is known through the
use of G-CSF receptor (G-CSFR)-deficient mice that the
G-CSFR is crucial for G-CSF-induced MPC mobilization
(24). Interestingly, no increase in circulating CFU-GM was
detected in G-CSFR
/
mice in the absence of added G-CSF
and after administration of IL-8 (24). Thus, CXC chemokine receptors have been linked to MPC mobilization by
G-CSF through a G-CSFR.
Although CCR1 has been implicated in MIP-1 suppression of BFU-E proliferation in vitro (23), our studies
suggest that CCR1 is not a dominant receptor for suppression of MPCs. We have previously shown no significant
difference in cycling of MPCs in CCR1
/
versus
CCR1+/+ marrow (20), consistent with CCR1 not being a
dominant receptor for negative regulation of MPC proliferation. This contrasts with studies in other chemokine receptor-deficient mice. CCR2 binds several chemokines,
including MCP-1 and its murine analogue, JE (25). The
use of CCR2
/
mice demonstrated that CCR2 was a
dominant receptor for suppression of MPCs by MCP-1
and JE (25). The relevance of CCR2 as a receptor involved
in negative regulation of MPCs was confirmed by the fact that the cycling status of MPCs in CCR2
/
marrow is
greater than that in CCR2+/+ marrow (26). IL-8 and
muMIP-2 bind CXCR2, and the use of CXCR2
/
mice
demonstrated that IL-8 and muMIP-2 did not inhibit proliferation of MPCs from CXCR2
/
marrow, and that enhanced proliferation of MPCs was apparent in CXCR2
/
compared with CXCR2+/+ mice (27).
In conclusion, our data identify CCR1 as the dominant
receptor responsible for MIP-1 enhancement of growth
factor-stimulated MPC proliferation and MIP-1
-induced
MPC mobilization to peripheral blood, but rule out CCR1
as a dominant receptor for negative regulation of hematopoiesis by MIP-1
. In addition, the results identify an unexpected role for CCR1 as a negative regulator of G-CSF- dependent MPC mobilization to blood. Together with
previous work, our results indicate that CCR1 is a highly
versatile receptor able to mediate a broad range of MIP-1
actions, including specific steps in MPC proliferation, development, and distribution, as well as specific leukocyte
trafficking. Delineation of specific downstream signaling
events will be needed to understand the molecular basis for
these diverse functional responses mediated by MIP-1
activation of CCR1.
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Footnotes |
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Address correspondence to Hal E. Broxmeyer, Walther Oncology Center, Indiana University School of Medicine, 1044 West Walnut St., Indianapolis, IN 46202-5254. Phone: 317-274-7510; Fax: 317-274-7592; E-mail: hbroxmey{at}iupui.edu
Received for publication 22 February 1999 and in revised form 23 April 1999.
We thank Cynthia Booth for typing this manuscript, and Stephen Braun and Jennifer Jayne for help with plating one set of experiments.
These studies were supported by U.S. Public Health Service grants R01 HL56416 and R01 DK53674 from the National Institutes of Health to H.E. Broxmeyer.
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