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Blood, 1 December 2001, Vol. 98, No. 12, pp. 3476-3478
BRIEF REPORT
Macrophage inflammatory protein-1 uses a novel receptor for
primitive hemopoietic cell inhibition
Katrin Ottersbach,
Donald
N. Cook,
William A. Kuziel,
Alison Humbles,
Bao Lu,
Craig Gerard,
Amanda E. I. Proudfoot, and
Gerard J. Graham
From the Beatson Institute for Cancer Research, CRC
Beatson Laboratories, Glasgow, Scotland; Schering-Plough Research
Institute, Kenilworth, NJ; Department of Molecular Genetics and
Microbiology and Institute for Cellular and Molecular Biology,
University of Texas, Austin; The Ina Sue Perlmutter Laboratory,
Childrens Hospital, Boston, MA; and Serono Pharmaceutical Research
Institute, Geneva, Switzerland.
 |
Abstract |
Macrophage inflammatory protein-1 (MIP-1) is a member of the
chemokine family of proinflammatory mediators. In addition to its
inflammatory roles, MIP-1 has been shown to be active as an
inhibitor of primitive hemopoietic cell proliferation. Indeed, a
dysfunction in this inhibitory process has been postulated to contribute to leukemogenesis. Research has been aimed at characterizing the receptor involved in cellular inhibition by MIP-1. This study demonstrates that of all the  -chemokines tested, only MIP-1 is
capable of inhibiting primitive hemopoietic cell proliferation. Because
no MIP-1-specific receptors have been identified, this suggests
that inhibition is mediated by an uncharacterized receptor. Further
evidence for the involvement of a novel receptor in this process is the
equivalent potencies of MIP-1S and MIP-1P variants of human
MIP-1 and the fact that primitive cells from bone marrow derived
from individual MIP-1 receptor null mice display a full response to
MIP-1 inhibition.
(Blood. 2001;98:3476-3478)
© 2001 by The American Society of Hematology.
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Introduction |
Chemokines are members of a large and expanding
family of proinflammatory mediators that is defined by the presence of
variations on a conserved Cys motif.1,2 There are
currently 4 chemokine subfamilies. The 2 most populous subfamilies have
4 Cys in their mature sequences and are respectively referred to as the
CC or -chemokine family and the CXC or -chemokine family. The
other 2 subfamilies are each represented by only single members, with the C family being represented by lymphotactin and the CXXXC family by
fractalkine/neurotactin. Chemokines have typically been characterized as proinflammatory mediators; however, we and others have demonstrated that chemokines, most notably macrophage inflammatory protein-1 (MIP-1), are active in inhibiting primitive hemopoietic cell proliferation in vitro and in vivo.3-5 Chemokines interact
with target cells via members of the 7 transmembrane family of
G-protein-coupled receptors.6 There is now a systematic
nomenclature for chemokine receptors, with -chemokines binding to
CCRs (CC chemokine receptor), -chemokines to CXCRs, and C or CX3C
chemokines to XCR and CX3CRs, respectively. To date, 11 CCRs, 6 CXCRs,
single XCR and CX3CR receptors, and 2 more promiscuous receptors (D6
and DARC) have been identified. MIP-1 binds to CCR1, CCR3 (in the
mouse), CCR5, and D6, but not to any of the other characterized
receptors.7,8
We have been attempting to characterize the receptor responsible for
inhibition of primitive murine hemopoietic cells by MIP-1. Identification of this receptor is of importance not only for enhancing
our understanding of the mechanisms of cellular inhibition by MIP-1,
but also potentially for unraveling aspects of the pathogenesis of a
number of leukemias, the primitive stem/progenitor cells that display a
dysfunction in their response to inhibition by
MIP-1.9-11 Whereas CCR1 null mice have been used to
demonstrate the lack of involvement of this receptor in inhibition of
primitive hemopoietic cells,12 there has been no
systematic examination of the involvement of all the known MIP-1
receptors in the inhibitory effects of this chemokine. Here we show,
using a range of chemokines, chemokine variants, and null mouse bone
marrow, that cellular inhibition by MIP-1 is not mediated through
any of the currently characterized receptors. We therefore believe that
inhibition of murine stem/progenitor cells by MIP-1 involves an
uncharacterized receptor.
| |
Study design |
Reagents
All chemokines were purchased from either R&D Systems Europe
(Oxford, United Kingdom) or PeproTech (London, United Kingdom), with the exception of murine MIP-113 and human
MIP-1P,14 which were generated in house.
AOP-RANTES was prepared as described previously.15
The colony-forming unit direct addition assay
Bone marrow cells were obtained by flushing from the femur and
either used immediately or frozen (for all experiments on receptor null
mice and their wild-type counterparts). Primitive hemopoietic cells
were assayed using the in vitro colony-forming unit-agar (CFU-A)
assay. This assay has been described in detail
elsewhere16,17 and detects a cell that is phenotypically
indistinguishable from day-12 spleen CFU cells. Briefly,
5 × 103 fresh bone marrow cells, or
5 × 104 defrosted bone marrow cells, were plated in 1 mL
0.3% agarose/25% donor horse serum (DHS) on top of a feeder layer
consisting of 0.6% agar/25% DHS/0.2 ng/mL recombinant murine
granulocyte macrophage colony-stimulating factor/6 ng/mL recombinant
human macrophage colony-stimulating factor, and 12 ng/mL stem cell
factor. Inhibition was assessed by directly adding the
chemokines to the assay plates and incorporating them into the feeder
layer.18 Assays were scored after 11 days, and CFU-A
colonies were identified as those with a diameter greater than 2 mm.
| |
Results and discussion |
To examine receptor usage, we initially investigated a variety of
-chemokines, representing ligands for each of the currently identified -chemokine receptors, for their ability to inhibit the
primitive CFU-A cells. As shown in Table
1, murine MIP-1 is fully active as an
inhibitor of CFU-A cell proliferation, with essentially complete
inhibition seen at a concentration of 50 ng/mL. In contrast, as shown
in Table 2, none of the other chemokines tested (at 100 ng/mL) displayed any consistent or significant inhibition of CFU-A colony formation. Thus, these data support a role
for MIP-1 as an inhibitor of primitive hemopoietic cells but
indicate that this activity is not shared with other -chemokines. The failure to demonstrate inhibition by chemokines other than MIP-1
is at odds with a number of reports demonstrating inhibition of
hemopoietic stem and progenitor cells with a wider range of chemokines,19,20 but is likely to be explained by
differences in the assays used in the different studies. Thus, the
CFU-A assay is ideally suited to the identification of the receptor
involved specifically in primitive cell inhibition by MIP-1. Because
there are currently no receptors identified that bind only MIP-1, it appears that MIP-1 inhibits CFU-A cell proliferation through an as
yet uncharacterized receptor.
We have recently characterized 2 nonallelic variants of human MIP-1
(MIP-1S and MIP-1P), both of which bind with similar affinities
to CCR1, but only one of which (MIP-1P) binds to murine CCR5
or D6.14 These variants allow us to assess the roles, if any, of CCR5 and D6 in the inhibitory process. In CFU-A assays, MIP-1P and MIP-1S display indistinguishable potencies, with half-maximal inhibition observed at approximately 30 ng/mL (Table 1),
further indicating that CCR5 and D6 are unlikely to be the inhibitory receptors.
The ultimate test of receptor involvement in a specific biologic
function is to examine cells from receptor null mice. To this end, we
examined the response of CCR1,21 CCR3 (Humbles et al,
manuscript in preparation), CCR5,22 and D6 (Cook et al, manuscript in preparation) null bone marrow cells to inhibition by
MIP-1. As shown in Table 3, all of the
null bone marrow samples displayed a full inhibitory response to
MIP-1, again indicating that none of the currently characterized
MIP-1 receptors is involved in CFU-A inhibition.
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Table 3.
The effects of MIP-1 and AOP-RANTES on percentage CFU-A
colony formation by wild-type and receptor null bone marrow
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It remains possible that MIP-1 can use a variety of chemokine
receptors to mediate inhibition of CFU-A cells. Thus, if MIP-1 can
use CCR1, CCR3, CCR5, or D6 for inhibition, the involvement of
an individual receptor would not be obvious in single-receptor null
mice. To test this possibility, we examined the inhibitory response of
CFU-A cells from individual-receptor null mouse bone marrow in the
presence of the chemokine variant AOP- RANTES.15,23 This
protein binds with high affinity to murine CCR1, CCR5 (Buser et al,
manuscript in preparation), and D6 (G.J.G., unpublished data, December
1997), but is inactive as an inhibitor of primitive hemopoietic cells at concentrations up to 500 ng/mL (data not shown).
Thus, for the purposes of the present study, AOP-RANTES may be regarded
as a blocker of these 3 receptors. As shown in Table 3, excess
AOP-RANTES had no effect on the ability of MIP-1 to inhibit CFU-A
cells from any of the receptor null bone marrow samples, suggesting
that in the absence of one specific receptor, MIP-1 is not using the
other known AOP-RANTES-sensitive receptors to mediate
inhibition. Although these data do not rigorously rule out the alternative use of CCR3, the full inhibitory response of the
CCR3 / cells, the inability of eotaxin to work as an
inhibitor, and the inability of excess eotaxin (20×) to block MIP-1
inhibition (G.J.G., unpublished observations, February 2000)
argue strongly against an involvement of this receptor in the
inhibitory process.
Thus, the above data are consistent with the use of a novel receptor
for inhibition of primitive hemopoietic cells by MIP-1. Although
these studies have necessarily been performed using murine bone marrow,
it is hoped that the results will have relevance to MIP-1 inhibition
of human primitive cells, a process we have demonstrated previously to
be independent of CCR1.24
| |
Footnotes |
Submitted February 5, 2001; accepted July 16, 2001.
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.
Reprints: Gerard J. Graham, Beatson Institute for Cancer
Research, CRC Beatson Laboratories, Garscube Estate, Switchback Rd,
Bearsden, Glasgow, G61 1BD, Scotland; e-mail:
g.graham{at}beatson.gla.ac.uk.
| |
References |
1.
Rollins BJ.
Chemokines.
Blood.
1987;90:909-928[Free Full Text].
2.
Zlotnik A, Yoshie O.
Chemokines: a new classification system and their role in immunity.
Immunity.
2000;12:121-127[CrossRef][Medline]
[Order article via Infotrieve].
3.
Graham GJ.
Growth inhibitors in haemopoiesis and leukaemogenesis.
Baillieres Clin Haematol.
1997;10:539-559[CrossRef][Medline]
[Order article via Infotrieve].
4.
Broxmeyer HE, Sherry B, Lu L, et al.
Enhancing and suppressing effects of recombinant murine macrophage inflammatory proteins on colony formation in vitro by bone marrow myeloid progenitor cells.
Blood.
1990;76:1110-1116[Abstract/Free Full Text].
5.
Broxmeyer HE, Kim CH, Cooper SH, Hangoc G, Hromas R, Pelus LM.
Effects of CC, CXC, C and CX3C chemokines on proliferation of myeloid progenitor cells and insights into SDF-1 induced chemotaxis of progenitors.
Ann N Y Acad Sci.
1999;872:142-163[CrossRef][Medline]
[Order article via Infotrieve].
6.
Murphy PM, Baggiolini M, Charo IF, et al.
International Union of Pharmacology, XXII: nomenclature for chemokine receptors.
Pharmacol Rev.
2000;52:145-176[Abstract/Free Full Text].
7.
Nibbs RJB, Wylie SM, Pragnell IB, Graham GJ.
Cloning and characterisation of a novel murine beta chemokine receptor, D6: comparison to three other related macrophage inflammatory protein-1 receptors.
J Biol Chem.
1997;272:12495-12504[Abstract/Free Full Text].
8.
Nibbs RJB, Wylie SM, Yang J, Landau NR, Graham GJ.
Cloning and characterisation of a novel promiscuous human -chemokine receptor D6.
J Biol Chem.
1997;272:32078-32083[Abstract/Free Full Text].
9.
Eaves CJ, Cashman JD, Wolpe SD, Eaves AC.
Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1, an inhibitor of primitive normal hematopoietic cells.
Proc Natl Acad Sci U S A.
1993;90:12015-12019[Abstract/Free Full Text].
10.
Owen-Lynch PJ, Adams JA, Brereton ML.
The effect of the chemokine rhMIP-1 and a non-aggregating variant BB10010 on blast cells from patients with acute myeloid leukaemia.
Br J Haematol.
1996;95:77-84[CrossRef][Medline]
[Order article via Infotrieve].
11.
Ferrajoli A, Talpaz M, Zipf TF.
Inhibition of acute myelogenous leukaemia progenitor proliferation by macrophage inflammatory protein 1.
Leukemia.
1994;8:798-805[Medline]
[Order article via Infotrieve].
12.
Gao JL, Wynn TA, Chang Y, et al.
Impaired host defense, hematopoiesis, granulomatous inflammation and type 1-type 2 cytokine balance in mice lacking CC chemokine receptor 1.
J Exp Med.
1997;185:1959-1968[Abstract/Free Full Text].
13.
Graham GJ, MacKenzie J, Lowe S, et al.
Aggregation of the chemokine MIP-1 is a dynamic and reversible phenomenon: biochemical and biological analyses.
J Biol Chem.
1994;269:4974-4978[Abstract/Free Full Text].
14.
Nibbs RJB, Yang J, Landau NR, Mao J-H, Graham GJ.
LD78, a non-allelic variant of human MIP-1 (LD78), has enhanced receptor interaction and potent HIV suppressive activity.
J Biol Chem.
1999;274:17478-17483[Abstract/Free Full Text].
15.
Simmons G, Clapham PR, Pigard L, et al.
Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist.
Science.
1997;276:276-279[Abstract/Free Full Text].
16.
Pragnell IB, Wright EG, Lorimore SA, et al.
The effect of stem cell proliferation regulators demonstrated with an in vitro assay.
Blood.
1988;72:196-201[Abstract/Free Full Text].
17.
Lorimore SA, Pragnell IB, Eckmann L, Wright EG.
Synergistic interactions allow colony formation in vitro by murine haemopoietic stem cells.
Leuk Res.
1990;14:481-489[CrossRef][Medline]
[Order article via Infotrieve].
18.
Graham GJ, Freshney MG.
In: Proudfoot AEI, Wells TNC, Power CA, eds. CFU-A assay for measurement of the antiproliferative effects of chemokines on murine early hemopoietic progenitors. Methods in Molecular Biology. Vol 138: chemokine protocols. Totowa, NJ: Humana Press; 2000:179-189.
19.
Broxmeyer HE, Kim CH.
Chemokines and hematopoiesis. In:
Rollins BJ, ed.
Chemokines and Cancer. Totowa, NJ: Humana Press; 1999:263-291.
20.
Patel VP, Kreider BL, Li Y, et al.
Molecular and functional characterisation of two novel human CC chemokines as inhibitors of two distinct classes of myeloid progenitors.
J Exp Med.
1997;185:1163-1172[Abstract/Free Full Text].
21.
Gerard C, Forssard JL, Bhatia M, et al.
Targeted disruption of the beta-chemokine receptor CCR1 protects against pancreatitis-associated lung injury.
J Clin Invest.
1997;100:2022-2027[Medline]
[Order article via Infotrieve].
22.
Kuziel W, Maeda N.
CCR5. In:
Mak TW, ed.
The Gene Knockout Factsbook. San Diego, CA: Academic Press; 1998:120-121.
23.
Elsner J, Mack M, Bruhl H, et al.
Differential activation of CC chemokine receptors by AOPRANTES.
J Biol Chem.
2000;275:7787-7794[Abstract/Free Full Text].
24.
Graham GJ, Wilkinson PC, Nibbs RJB, et al.
Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1 by mutagenesis of the proteoglycan binding site.
EMBO J.
1996;15:6506-6515[Medline]
[Order article via Infotrieve].
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Abstract
Introduction