Multiple myeloma (MM) represents a neoplastic B-cell disorder, characterised by a
monoclonal and uncontrolled expansion of malignant plasma cells within the bone marrow
(BM) compartment (Ludwig et al, 1999). Although MM cells primarily localise in the
BM, they can also be found in the circulation, and the number of circulating cells
increases at very advanced stages of disease. It can be assumed that these circulating
MM cells are involved in disease spreading and must therefore have the potential to
extravasate and home to the BM (Van Riet et al, 1998). In analogy to the migration
and homing of normal lymphocytes, the BM homing of MM cells is likely to be mediated
by specific mechanisms, involving the action of locally produced factors with chemotactic
properties (Butcher and Picker, 1996). Chemokines are a family of small, structurally
related cytokines with chemoattractant and activation properties involved in several
types of inflammatory reactions (Oppenheim et al, 1991). They are characterised by
the presence of a conserved cysteine motif near the N-terminal end. They can be classified
into four distinct groups (C, CC, CXC and CX3C chemokines) based on the presence or
absence of an amino-acid sequence separating the two first cysteines (Baggiolini et
al, 1994; Premack and Shall, 1996). They are produced by a number of cell types, including
leukocytes, endothelial cells, fibroblasts and stromal cells. Chemokines act on responsive
cell types through G-protein coupled seven transmembrane receptors (Murphy et al,
1994). They have primarily been functionally related to leukocyte trafficking, but
recent reports suggest their role in cancer development and progression as well (Strieter,
2001; Muller et al, 2001).
CCR2 is a chemokine receptor that is expressed on peripheral blood monocytes, as well
as activated T cells, B cells and immature dendritic cells (Frade et al, 1997; Vecchi
et al, 1999). Gene-targeted mice lacking CCR2 (CCR2−/− mice) exhibit defects in monocyte/macrophage
trafficking to sites of inflammation (Kurihara et al, 1997; Boring et al, 1998; Peters
et al, 2000). The known ligands for CCR2 include the monocyte chemotactic proteins
(MCPs) MCP-1, -2 and -3 belonging to the family of CC chemokines (Mellado et al, 1998).
They act as potent activators and chemoattractants for monocytes, basophils, eosinophils,
T-lymphocyte subsets, dendritic cells and endothelial cells, but not neutrophils (Baggiolini
et al, 1994; Salcedo et al, 2000). In addition, MCP-1 and -3 have shown antitumour
activity by chemokine gene transfer in mouse models (Hoshino et al, 1995; Fioretti
et al, 1998). MCP-1 has also been implicated in angiogenesis (Salcedo et al, 2000).
To date, very few data are available about the effects of chemokines on human MM cell
migration (Woodliff and Epstein, 1999,2000). In this study, we analysed the functional
expression of CCR2 on MM cell lines (HMCL) as well as primary MM cells from BM of
MM patients. Our data demonstrate that HMCL, as well as primary MM cells, express
CCR2. In addition, the MCPs, produced by BM stromal cells, felicitated migration responses,
suggesting a potential contribution to the homing of MM cells to the BM microenvironment.
MATERIALS AND METHODS
Chemokines and antibodies
The human recombinant monocyte chemotactic proteins MCP-1, -2 and -3 were obtained
from Biosource. Monoclonal antibodies (MoAb) against MCP-1, -2 and -3 were purchased
from R&D. The CCR2 MoAb was a kind gift from Dr C Clement (Millennium Pharmaceuticals,
Cambridge, MA, USA).
Cell lines
Three well-characterised human MM cell lines (HMCL) (LP-1, Karpas and MM5.1) were
selected for our experiments. They were kept in culture as described (Nilson, 1971;
Okuno et al, 1991; Van Riet et al, 1997).
Patient samples
BM samples from 28 MM patients (pts) (age 41–94 years, mean 66) were collected during
standard diagnostic procedures. Each MM patient was diagnosed and staged according
to the criteria of Durie and Salmon (1975). The study was approved by the local ethical
committee. BM aspirates were obtained from the posterior iliac crest or sternum and
collected in a heparinised syringe. Mononuclear cells (MNC) were separated by Ficoll
density gradient centrifugation (Nycomed, Lucron, Gent, Belgium).
MACS separation of primary MM cells
Primary MM cells were immunomagnetically separated using the magnetic cell sorting
system (MACS) (Miltenyi Biotech Sanvertech, Bouchout, Belgium). MNC were incubated
for 15 min at 4°C with MACS microbeads conjugated to a monoclonal mouse CD138 (syndecan-1)
antibody (clone B-B4, isotype mouse IgG1). Cells were washed once in PBS supplemented
with human albumin (4%), resuspended and separated on a column placed in the magnetic
field of the MACS separator. CD138+ cells were retained and eluted as a positively
selected cell fraction after removal of the column from the magnetic field. Cells
were counted and viability was assessed with trypan blue. MACS purification produced
a 98% pure primary MM cell population as determined by May – Grünwald – Giemsa-stained
cytospin preparation.
BM stromal cell culture and conditioned medium
MNC from BM samples obtained from MM patients and normal controls were cultured in
75 cm2 flasks (Nunc, VWR International, Leuven, Belgium) in RPMI supplemented with
12.5% foetal calf serum (FCS) and 12.5% horse serum at 37°C with 5% CO2. After 3–5
weeks of culture, a confluent layer was obtained and stromal cells were detached by
trypsinisation. After one passage, established confluent cell layers were cultured
for 5 days in serum-free medium (RPMI) and culture supernatant was harvested as conditioned
medium (CM). CM was centrifuged to remove cell debris and frozen at −20°C until use.
CM preparations from different cell cultures were prepared.
RNA extraction, cDNA synthesis and reverse transcription polymerase chain reaction
(RT–PCR)
Total RNA was extracted from cultured HMCL and cultured BM stromal cells from normal
and MM BM samples using the RNeasy Mini Kit (Qiagen), according to the manufacturer's
instructions. First-strand cDNA was generated from 5 μg of total RNA with the SUPERSCRIPT™
Preamplification System (GIBCO BRL) according to the instructions made by the manufacturer.
The amount of DNA corresponding to 1/10 of the cDNA obtained by reverse transcription
was amplified with a PCR protocol in the presence of HotStarTaq Master Mix (Qiagen)
and primer pairs (10 μ
M) (GIBCO BRL) in a 25 μl reaction mixture. The following primers were used for amplifying
mRNA: CCR2 sense: 5′-TGG CTG TGT TTG CTT CTG TC-3′ and CCR2 antisense: 5′-TCT CAC
TGC CCT ATG CCT CT-3′, (actin sense: 5′-TGC CTA TCC AGG CTG TGC TAT-3′ and actin antisense:
5′-GAT GGA GTT GAA GGT AGT TT-3′; MCP-1 sense: 5′-CTC AGC CAG ATG CAA TCA ATG C-3′
and MCP-1 antisense: 5′-CCT CAA GTC TTC GGA GTT TGG G-3′; MCP-2 sense: 5′-ATG CTG
AAG CTC ACA CCC TTG CCC-3′ and MCP-2 antisense: 5′-CAG ATG CTT CAT GGA ATC CCT GAC
C-3′; MCP-3 sense: 5′-CAG ATT TAT CAA TAA GAA AAT CCC-3′ and MCP-3 antisense: 5′-GTG
CTT CAT AAA GTC CTG GAC CC-3′ (Dumoulin et al, 1999). The PCR profile consisted of
an initial activation step of 15 min at 95°C, a 1 min initial denaturation at 94°C,
followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 58, 55, 58,
68 and 54°C for CCR2, actin, MCP-1, -2 and -3, respectively, 2 min renaturation at
72°C and finally 10 min extension at 72°C. PCR products were analysed by electrophoresis
in a 2% agarose gel, visualised by ethidium bromide and photographed (Kodak EDAS 290).
The amplified mRNA was identified based upon the anticipated size by comparison with
DNA ladder of known molecular sizes.
Flow cytometry
Flow cytometry was used to assess CCR2 expression on the surface of HMCL and primary
MM cells. For phenotyping the HMCL, cells were first incubated with mouse anti-human
CCR2 MoAb (IgG2a) or control mouse IgG2a (both at 10 μg ml−1) for 30 min at 4°C. In
the second step, cells were incubated with PE-conjugated goat anti-mouse IgG2a antiserum
(Southern Biotechnology InTec, Antwerpen, Belgium) for 30 min at 4°C. Cells were washed,
resuspended in PBS and analysed on EPICS XL flow cytometer (Coulter Electronics Analis,
Namur, Belgium). CCR2 expression on the surface of primary MM cells and normal plasma
cells was evaluated by a double-staining procedure. MNC, isolated from BM samples
of MM patients and normal controls by Ficoll gradient centrifugation, were incubated
with CCR2 MoAb as described and a Cy-5-conjugated CD38 specific antibody (HIT2) (Becton
Dickinson Erembodgem, Belgium). In the second step, cells were incubated with PE-conjugated
goat anti-mouse IgG2a antiserum (Southern Biotechnology). The fluorescence intensity
was calculated from the fluorescence histogram and expressed as the fluorescence intensity
ratio (FiR) (specific fluorescence/control fluorescence). A FiR value of more than
1.6 was considered positive.
In vitro cell migration assay
Migration of HMCL and primary MM cells was assayed using Transwell™ cell culture inserts
(Costar Corning Elscolab, Kruibekes, Belgium) as described previously (Vande Broek
et al, 2001). CM from BM stromal cells or recombinant chemokines MCP-1, -2 and -3,
diluted in 300 μl RPMI 1640 medium in varying concentrations, were placed into 24-well
culture plates (Costar Corning). Transwell™ inserts (6.5 mm diameter, 8 μm pore size)
were placed in each well and 1 × 105 MM cells in 100 μl RPMI were added to the upper
chamber. Cells were allowed to migrate for 4 h at 37°C with 5% CO2, after which inserts
were removed. The number of cells that transmigrated into lower wells was evaluated
in two ways. For HMCL, a colorimetric assay with WST-8 was used (Cell Counting Kit-8
(Alexis)). WST-8 was added in each well and incubated for 4 h at 37°C. The absorbance
of converted stain was then measured spectrophotometrically with a 96-well microplate
reader (Ceres 900, Bio-Tek International Inc, Brussels, Belgium) at a wavelength of
450 nm with a reference wavelength of 620 nm. For primary MM cells, transmigrated
cells were recovered from lower wells and counted with a FACSort flow cytometer (Becton
Dickinson Erembodgem, Belgium). A known number of sphero blank calibration beads (Becton
Dickinson Erembodgem, Belgium) were used as internal standard. Experiments were performed
in triplicate and the mean with standard deviations (s.d.) was calculated. Migration
responses were determined as the mean increase in cell migration as compared to control
(spontaneous) migration. For migration-inhibition experiments, cells were preincubated
for 30 min with a blocking CCR2 MoAb. In some experiments, cell migration was assayed
in the presence of neutralising MoAbs against MCP-1, -2 and -3, which had been added
to lower wells.
RESULTS
CCR2 is expressed by human MM cell lines (HMCL) and primary MM cells from patient
BM samples
We first determined the expression of CCR2 by HMCL using RT – PCR and flow cytometry.
RT – PCR of MM cell mRNA with specific primers for CCR2 showed the presence of CCR2
transcripts in all three HMCL tested (Figure 1A
Figure 1
Expression of CCR2 chemokine receptor (A and B) and in vitro migration to MCP-1, -2
and -3 (C) by HMCL. CCR2 expression on HMCL was analysed by RT–PCR (A) and flow cytometry
(B). RT–PCR analysis from total RNA extracted from HMCL with CCR2-specific primers
showed a specific 230 bp PCR fragment in all three HMCL tested. Analysis of actin
mRNA expression (446 bp PCR fragment) served as an internal control. Water was used
as negative control. FACS analysis showed surface expression of CCR2 on HMCL. Results
are shown as fluorescence histograms (open histogram: CCR2 expression; filled histogram:
isotype-matched control antibody). Three HMCL were tested for their ability to migrate
across 8 μm pore-size polycarbonate filters in response to different MCP-chemokine
concentrations as indicated (C). Values are expressed as the percentage increase of
control migration to serum-free medium. Results shown are the mean and s.d. of three
independent experiments.
). Flow cytometry with CCR2 MoAb demonstrated surface expression of CCR2 in all three
HMCL (Figure 1B). Subsequently, we analysed surface expression of CCR2 on primary
MM cells in BM samples from MM patients and normal controls using a two-colour staining
method. FACS profiles revealed that CCR2 was expressed on primary MM cells in the
majority (82%) of the patient samples tested (n=28). Results of CCR2 expression in
primary MM cells from four BM samples are illustrated in Figure 2A
Figure 2
Expression and functionality of CCR2 on primary MM cells from BM samples: (A) FACS
analysis of CCR2 on primary MM cells: Results are shown as fluorescence histograms.
The open histogram shows CCR2 expression; the filled histogram represents the isotype-matched
control antibody. Representative results of CCR2 expression from four different BM
samples are shown. (B) In vitro cell migration to MCP-1, -2 and -3 was assayed using
immunomagnetically isolated primary MM cells from four patient BM samples. Chemokines
were used at 100 ng ml−1. The number of migrated cells was quantified by flow cytometry.
Data represent the percentage increase of control migration. Representative values
are shown for experiments with primary MM cells from four patient samples.
. Among the positive cases, the mean fluorescence intensity varied between 1.6 and
14.4. CCR2 was also found to be expressed on plasma cells from all normal BM samples
tested (n=10) (data not shown).
MCP-1, -2 and -3 act as chemoattractants for HMCL and primary MM cells
Since HMCL and primary MM cells express CCR2, the major chemokine receptor for the
MCPs, it can be assumed that MCP-1, -2 and -3 function as chemoattractants for human
MM cells. Therefore, we evaluated the migration of HMCL and primary MM cells in the
presence of MCP-1, -2 and -3. Addition of MCP-1, -2 and -3 at concentrations from
1 to 1000 ng ml−1 to the lower compartments of the migration system resulted in a
concentration-dependent stimulation of MM cell migration (Figure 1C). In the three
HMCL, the maximal increase in cell migration to MCP-1 ranged between 38 and 75%, corresponding
with 21–28% of the total number of cells in the upper compartment that actively migrated
through the membrane into the lower compartment of the Transwell migration system.
For MCP-2, the maximal increase in migration observed, varied between 41 and 56%,
corresponding with 25–28% of the total number of input cells that migrated through
the filter. In the presence of MCP-3, the maximal increase in migration observed,
ranged between 54 and 68%, corresponding with 27–30% of the cells migrating to the
lower compartment. Similar results were obtained using isolated primary MM cells from
three MM patients (Pts 1 – 3), which were positive for CCR2. For all these patients,
the most pronounced migration response was observed with MCP-1. In the presence of
this chemokine, we observed an increase in cell migration between 48 and 60%, corresponding
with 29–48% of cells migrating to the lower compartment. In one MM patient with CCR2-negative
plasma cells (Pt 4), no significant migration response towards MCP-1, -2 or -3 could
be observed (Figure 2).
BM stromal cells express mRNA for MCP-1, -2 and -3
Using specific primers for MCP-1, -2 and -3, we amplified PCR products of expected
sizes (372, 300 and 160 bp, respectively) from cDNA of stromal cells, cultured from
normal and MM BM samples (Figure 3
Figure 3
Expression of MCP-1, -2 and -3 in human BM stroma from MM patients and normal controls.
Total RNA was isolated from cultured human BM stromal cells from MM patients and normal
controls, and subjected to reverse transcription and PCR amplification for MCP-1,
-2 and -3 using appropriate primers. The specific 372, 300 and 160 bp PCR fragments
for MCP-1, -2 and -3, respectively, were detected in all normal and MM BM samples
tested. Analysis of actin mRNA expression (446 bp PCR fragment) served as an internal
control. Water was used as negative control.
). Analysis of actin mRNA expression served as an internal control. These findings
indicate that BM stromal cells from MM BM samples and normal controls produce the
chemokines MCP-1, -2 and -3.
Conditioned medium (CM) from BM stromal cells stimulates MM cell migration
We next determined whether supernatants from cultured BM stromal cells could stimulate
MM cell migration. Migration of HMCL and primary MM cells was determined as described
in Materials and Methods. CM was placed in the lower compartments of the migration
system. As shown in Figure 4A
Figure 4
Effect of CM from BM stromal cells on human MM cell migration and inhibition by anti-CCR2
MoAb. In vitro cell migration was analysed by in vitro Transwell migration assay.
BM CM was added to the lower compartment. Migration of three HMCL (Karpas, MM5.1 and
LP-1) and primary MM cells from four MM patient samples was assayed. Results are expressed
as the percentage increase of control migration to serum-free medium (A). For migration-inhibition
experiments, MM cells were preincubated with a MoAb against the chemokine receptor
CCR2 or a control antibody (IgG2a) prior to the migration assay. Results indicate
the relative migration compared with control migration to CM and represent the mean
value±s.d. of three experiments with Karpas cells. Representative values are also
shown for experiments with primary MM cells from three MM patients (B).
, the migration response of HMCL and primary MM cells was significantly enhanced (67
– 83 and 52 – 89% respectively, corresponding with 25 – 28 and 38 – 41% of migrating
cells, respectively) when compared to control migration to serum-free RPMI medium.
No differences were found in migration responses to CM from stromal cells, cultured
from normal BM samples as compared to CM from MM BM samples (data not shown).
CM-induced MM cell migration is inhibited by MoAbs against CCR2 and MCP-1, -2 and
-3
To determine whether the chemokines MCP-1, -2 and -3 produced by BM stromal cells
are involved in the chemoattractive effect of BM CM, we performed migration experiments
with MM cells towards BM CM in the presence of a blocking CCR2 MoAb or neutralising
MCP MoAbs. MM cells were preincubated with CCR2 MoAb prior to the migration assay,
and migration responses towards BM CM were analysed. In some experiments, MCP-1, -2
and -3 MoAbs were added to the lower compartment of the migration system together
with CM. MM cell migration to BM was significantly inhibited by CCR2 MoAb up to 40%
for Karpas cells and up to 52% for the patient samples (Figure 4B). The presence of
one single MoAb against MCP-1, -2 or -3 did not significantly affect MM cell migration
to BM CM, but in the presence of three MoAbs together against MCP-1, -2 and -3, MM
cell migration was reduced up to 40% for Karpas cells and up to 50% for the isolated
primary MM cells (Figure 5A,B
Figure 5
In vitro MM cell migration to CM is inhibited by anti-MCP MoAb. MoAb against MCP-1,
-2 and -3 or control antibody (IgG1) was added together with CM to the lower compartment
of the migration system. Results indicate the relative migration compared with control
migration to CM and represent the mean value±s.d. of three experiments with Karpas
cells (A). Representative values are shown for experiments with primary MM cells from
three MM patients (B).
). These experiments clearly demonstrate that CM-induced MM cell migration involves
MCP-1, -2 and -3 and that MM cell migration to MCPs occurs through CCR2.
DISCUSSION
A striking feature of MM represents the selective localisation of malignant plasma
cells in the BM. The mechanisms by which MM cells traffic to and accumulate in the
BM are not fully understood. In analogy to the migration and homing of normal lymphocytes
(Butcher and Picker, 1996), one can hypothesise that the BM homing of MM cells is
mediated by a multistep process. First, circulating cells reversibly roll onto the
endothelium, followed by a firm adhesion, transendothelial migration with passing
through the basement membrane and finally migration to the extracellular matrix. Our
group has demonstrated that murine 5T2 MM cells selectively migrate to the BM, the
spleen and the liver, but only survive within the BM compartment and the spleen. So,
the selective localisation of MM cells in the BM likely results from a combination
of a selective homing and survival in the BM (Vanderkerken et al, 2000). In the same
murine 5T model, we could also recently demonstrate that the BM homing of MM involves
the adhesion receptor CD44v6 that mediates binding to BM endothelium (Asosingh et
al, 2000). It is believed that homing not only requires specific cellular adhesion,
but also depends on the interaction of locally produced chemoattractants with specific
cell surface receptors (Butcher and Picker, 1996). Previously, we have demonstrated
that laminin-1, a major component of the basement membrane, acts as a potent chemoattractant
for human MM cells, indicating a key role for extravasation through the basement membrane
(Vande Broek et al, 2001). In addition, MM cells were found to express the high-affinity
laminin binding protein 67LR, which appeared to be upregulated by contact with the
endothelium, indicating that during passage through the basement membrane, 67LR is
contemporary upregulated allowing MM cells to be sensible to the chemoattractive properties
of laminin-1, and so facilitating transendothelial migration. As a result of the broad
expression of laminin-1 in basement membranes throughout the body, it is clear that
this molecule on itself cannot be the only factor that determines the specificity
of MM cell homing to the BM. We therefore focussed on other molecules with chemoattractive
potential and started to analyse the particular role of chemokines in the BM homing
process of MM cells. Chemokines represent a family of growing interest and recent
findings indicate that these molecules are implicated in a complex network of signalisation
between tumour cells and the microenvironment of the host (Amann et al, 1998; Ferrero
et al, 1998).
We report here that HMCL and primary MM cells freshly isolated from the BM of MM patients
express the chemokine receptor CCR2, as analysed by RT–PCR and/or flow cytometry.
The chemokine receptor CCR2, together with CCR5, plays an important role in the recruitment
of monocytes/macrophages and T cells in various inflammatory diseases, infection and
arteriosclerosis (Kurihara et al, 1997; Boring et al, 1998). Previous reports have
demonstrated that CCR2 is expressed on normal, mature B cells (Frade et al, 1997).
Very recently, transcripts for this receptor were also identified in myeloma plasma
cells, as demonstrated by cDNA arrays (De Vos et al, 2001). Our data show that CCR2
is also expressed in both, normal and malignant plasma cells, at the protein level.
To our knowledge, no reports have been published yet describing the expression of
the chemokine receptor CCR2 in other B-cell malignancies. Using Transwell™ migration
assays, we could demonstrate that CCR2 expression on MM cells was functional since
ligands of CCR2, that is MCP-1 as well as MCP-2 and -3, act as chemoattractants for
human MM cells. Compared to control migration in the absence of chemoattractant, we
noticed an increased cell migration response up to 80% for MCP-1, 60% for MCP-2 and
70% for MCP-3.
To investigate whether CCR2 and MCP-1, -2 and -3 are directly involved in the BM homing
of MM cells, we first analysed the production of MCP-1, -2 and -3 by human stromal
cells, cultured from normal and MM BM samples. A previous report described the production
of MCP-1 by murine BM stromal cells (Xu et al, 1999). Moreover, MCP-2 mRNA has been
identified in human BM stromal cells (Van Coillie et al, 1997). In this study, we
demonstrated by RT–PCR, that BM stromal cells in MM samples express transcripts for
MCP-1, -2 and -3. To determine the role of these chemokines in mediating MM cell migration
to CM from cultured BM stromal cells, we performed in vitro migration assays in the
presence of a blocking CCR2 MoAb. A reduction in cell migration up to 52% was observed.
Additionally, in vitro migration assays were performed in the presence of neutralising
MoAbs against MCP-1, -2 and -3. Interestingly, we observed no reduction of cell migration
in the presence of one single MoAb or a combination of two MoAbs, but a clear reduction
in MM cell migration was found when the three chemokines were neutralised simultaneously.
It seems that preventing the binding of two MCP molecules to CCR2 still allows the
third MCP to elucidate migration responses. Inhibition of the migration response to
CM from BM stromal cells with a blocking CCR2 MoAb and neutralising MCP MoAbs indicates
that MCP-1, -2 and -3 represent a major chemotactic activity for MM cells released
by BM stroma. Moreover, the recent finding that IL-6, a major growth factor in MM,
can upregulate the production of MCP-1 (Biswas et al, 1998) suggests that at least
one of these chemokines is abundantly present in the BM microenvironment of MM patients.
The fact that blocking of CCR2 binding to MCP-1, -2 and -3 does not induce complete
inhibition of MM cell migration suggests that one or more additional chemoattractant(s)
is (are) involved in the BM homing of MM cells. Recent data indicate that human MM
cells express the chemokine receptor CXCR4 and that this receptor mediates in vitro
migration of MM cells to SDF-1, a BM stromal-derived factor (Woodliff and Epstein,
1999). Moreover, it was found that CXCR4-positive, but not CXCR4-negative, MM cell
lines home to the BM in SCID mice, suggesting the involvement of this particular receptor
in the BM homing of MM cells as well (Woodliff et al, 2000). Another chemokine that
might influence the migration behaviour of MM cells is MIP-1α. This chemokine was
found to be secreted at elevated levels by BM cells of MM patients and has been related
to osteoclast activation (Choi et al, 2000). Future experiments have to explore at
which level SDF-1 and MIP-1α co-act with the MCPs to mediate the chemoattraction of
MM cells to the BM.
The fact that CCR2 was also found to be expressed by normal plasma cells and its ligands
were found to be secreted by normal stromal cells as well indicates that these receptor–ligand
interactions might be involved in normal plasma cell homing as well. One study described
that CCR2−/− mice do not show obvious haematological abnormalities, but the distribution
and BM homing of normal plasma cells was not evaluated (Kurihara et al, 1997).
In conclusion, the results obtained in this study indicate that MM cells express CCR2
and that this chemokine receptor is functional. In addition, the monocyte chemotactic
proteins MCP-1, -2 and -3, produced by BM stroma, act as chemoattractants for human
MM cells and are involved in MM cell migration to BM stroma. These findings indicate
a contribution of the chemokine receptor CCR2 to the homing of MM cells to the BM
microenvironment.