Expression and function of chemokine receptors in human multiple ...

Leukemia (2003) 17, 203–210  2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

Expression and function of chemokine receptors in human multiple myeloma C Mo¨ller, T Stro¨mberg, M Juremalm, K Nilsson and G Nilsson Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden

Multiple myeloma (MM) is a B cell tumor characterized by its selective localization in the bone marrow. The mechanisms that contribute to the multiple myeloma cell recruitment to the bone marrow microenvironment are not well understood. Chemokines play a central role for lymphocyte trafficking and homing. In this study we have investigated expression and functional importance of chemokine receptors in MM-derived cell lines and primary MM cells. We found that MM cell lines express functional CCR1, CXCR3 and CXCR4 receptors, and some also CCR6. Although only a minority of the cell lines responded by calcium mobilization after agonist stimulation, a migratory response to the CCR1 ligands RANTES and MIP-1␣ was obtained in 5/6 and 4/6, respectively, of the cell lines tested. Five out of six cell lines showed a response to the CXCR4 ligand SDF-1. In addition, 3/6 cell lines migrated in response to MIP-3␣ and IP-10, ligands for CCR6 and CXCR3, respectively. The expression of CXCR4 and CCR1 and the migration to their ligands, SDF-1, and RANTES and MIP-1␣, respectively, were also demonstrated in primary MM cells. These findings suggest that chemokine receptor expression and the migratory capacity of MM cells to their ligands are relevant for the compartmentalization of MM cells in the bone marrow. Leukemia (2003) 17, 203–210. doi:10.1038/sj.leu.2402717 Keywords: multiple myeloma; migration; CCR1; CXCR4; RANTES; MIP-1␣; SDF-1

Introduction Multiple myeloma (MM) is a monoclonal expansion of malignant cells with a plasmablast–plasma cell morphology that is almost exclusively localized to the bone marrow. MM cells appear to be derived from a post germinal centre B cell as they express somatically mutated Ig heavy chain genes.1 The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. It is possible that the bone marrow localization reflects the expression of chemotactic receptors on MM cells that directs their migration to the bone marrow. Chemokines are a superfamily of cytokines that play a critical role in the selective recruitment and homing of leukocytes by acting as chemotaxins.2 The chemokines can be classified into four subfamilies, C, CC, CXC and C(X)3C, based on the number and arrangement of conserved cysteines.3 Today more than 40 different chemokines have been described, of which most belong to either the CC or CXC families. The chemokines mediate their effects by binding to seven transmembrane-spanning, G-protein coupled receptors. The role of chemotactic factors in directing migration of granulocytes, macrophages, and T lymphocytes has been extensively investigated, but less is known about B lymphocyte chemoattractants and their receptors.4,5 B cells at distinct differentiation stages are differentially localized in primary lymphoid tissues or bone marrow, probably due to their expression of chemokine receptors, their responsiveness to Correspondence: G Nilsson, Laboratory of Tumor Biology, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden; Fax: +46 18 558931 Received 23 October 2001; accepted 1 July 2002

chemokines, and to their pattern of expression of adhesion molecules.6 The chemokine receptors implicated in B cell migration and proliferation include CXCR4, CXCR5, CCR2, CCR6 and CCR7.7–14 The chemokine stromal cell-derived factor-1 (SDF-1) and its corresponding receptor CXCR4 have been shown to be essential for bone marrow myelopoiesis and B lymphopoiesis.15,16 SDF-1 is constitutively expressed at high levels by bone marrow stromal cells.17,18 CXCR4 appears to participate in the regulation of B lymphopoiesis by confining precursors within the bone marrow microenvironment.19 The chemotactic responsiveness of B cells to SDF-1 appears to be related to their differentiation stages, where mature forms of B cells although still expressing CXCR4, do not migrate towards SDF1.7,20–22 Human MM cells have been demonstrated to express CXCR4, but the functionality of these receptors has not been investigated previously.23 Here, we systematically investigated the expression of chemokine receptors in MM cell lines. The functionality of receptors was tested by agonist-induced calcium mobilization and cell migration. We also investigated functional expression of CXCR4 and CCR1 in primary MM cells. Materials and methods

Cells A panel of human MM cell lines was used for the study: U266 1970,24 U-266 1984,25 U-1958,26 Karpas 707,27 LP-1,28 L-363,29 HL407E and HL407L.30 All the cell lines are negative for EBV, express CD138, and are classified as true MM cell lines.31 The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 50 ␮g/ml streptomycin (Life Technologies, Renfrewshire, UK). U-266 1970, U-1958, and HL407E are IL-6dependent cell lines and were grown on a layer of IL-6-producing human fibroblast lines AG1523 (The Human Mutant Genetic Cell Repository, Camden, NJ, USA). Medium was replenished twice a week.

Purification of primary MM cells Bone marrow samples were obtained from patients with newly diagnosed MM. The use of fresh human tissue was approved by the local ethical committee according to the Helsinki Declaration and MM BM cells were obtained after informed consent. Mononuclear cells were purified by centrifugation over Ficoll–Hypaque density gradient (Pharmacia, Uppsala, Sweden), washed and incubated with anti-CD138coated microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA, USA). Positive selection of magnetically labelled cells using an LS column and MidiMACS resulted in a purity of ⬎98% plasma cells as judged by morphological examination of MGG-stained cytospin preparations.

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Analysis of RNA expression by RNase protection assay and RT-PCR Total RNA was prepared using the TriPure Isolation Reagent (Boehringer Mannheim, Mannheim, Germany). Expression of chemokine receptor mRNA was analyzed by RNase Protection Assay (PharMingen, San Diego, CA, USA), with hCR5 and hCR6 multi-probe sets specific for CCR and CXCR, according to the manufacturer’s protocol and as described.32 In addition to RPA analysis we also performed RT-PCR analysis. The RNA was first reverse-transcribed with First Strand cDNA Synthesis Kit for RT-PCR (Boehringer Mannheim) according to the manufacturer’s protocol. PCR Core Kit (Boehringer Mannheim) was used for the amplification reactions according to the manufacturer’s protocol. mRNA from a pool of resting and activated (Con A 3 ␮g/ml or LPS 20 ␮g/ml) peripheral blood mononuclear cells

(PBMNC) was used as a positive control for both RPA and RT-PCR.

Flow cytometry analysis of chemokine receptor expression The MM-derived cell lines Karpas 707, U-1958, U-266 1970, U-266 1984, LP-1 and L363, and primary MM cells were analyzed by flow cytometry for cell surface expression of chemokine receptors as described.32 The mAbs used were: antihuman CXCR4-PE, anti-human CCR2-PE, anti-human CCR6PE, anti-human CXCR3-FITC (PharMingen), anti-human CCR1 and anti-human CCR1-FITC (R&D Systems, MN, USA). When unlabelled mouse anti-human CCR1 mAb were used, a secondary PE conjugated F (ab′)2 fragment of rabbit anti-mouse immunoglobulins (DAKO, Glostrup, Denmark) was added. Isotype-matched mouse antibodies IgG1 (DAKO), IgG2A (DAKO) and IgG2B (The Binding Site, Birmingham, UK) were used to detect non-specific background fluorescence.

Cytosolic calcium Changes in the intracellular calcium concentration ([Ca2+]i) were measured with the fluorescent indicator Fura-2AM (Molecular Probes, Eugene, OR, USA). Cells were washed with PBS and resuspended at 5 × 106 cells/ml in Hanks’ balanced salt solution w/o phenol red (HBSS) (Life Technologies) supplemented with 1% FBS. The cells were loaded with Fura2 AM (2.5 ␮g/ml) for 60 min at 30°C in the dark. The cells were then washed twice in HBSS supplemented with 1% FBS and resuspended at 1 × 106 cells/ml in the same medium. Three hundred and fifty ␮l cell suspension was placed in a magnetically stirred and thermostated quartz cuvette. [Ca2+]i was measured using excitation at 340/380 nm in a dual-wavelength fluorescence spectrophotometer (Hitachi-F2000, Toyo, Japan) after addition of the chemokines SDF-1␣, IP-10, MIP3␣, MCP-1, MIP-1␣ and RANTES (10–1000 ng/ml) (PeproTech, London, UK). Digitonin (Calbiochem-Novabiochem, Bad Soden, Germany) (60 ␮g/ml) and EGTA (Sigma, St Louis, MO, USA) (10 mM) were added to get maximal (Fmax) and minimal (Fmin) fluorescence values.

Migration assay

Figure 1 Expression of chemokine receptor mRNA in MM cell lines. Expression of CXCR (a) and CCR (b) was analyzed by RPA. Lanes: 1, probe; 2, L363; 3, LP-1; 4, K.707; 5, HL407L; 6, HL407E; 7, U-1958; 8, U-266 1984; 9, U-266 1970; 10, human control RNA; 11, PBMNC. Leukemia

Migration studies were performed using the disposable 96well chemotaxis chamber (ChemoTx; Neuroprobe, Gaithersburg, MD, USA)33 with a polycarbonate filter with a pore size of 5 ␮m and a filter width/well of 6 mm. The cells were resuspended at 1 × 106 cells/ml in PBS and loaded with Calcein AM (Molecular Probes) (1␮M) for 45 min at 37°C, 5% CO2. Following the incubation the cells were washed three times and resuspended in RPMI w/o phenol red (Life Technologies) supplemented with 0.5% BSA (Sigma) and 2 mM L-glutamine, 100 U/ml penicillin and 50 ␮g/ml streptomycin. Attractants to be tested were diluted in the medium mentioned above. Twenty-nine ␮l of the attractants were added to the wells at a concentration of 10, 100 and 500 ng/ml, and 50 ␮l of the cell suspension (2 × 106 cells/ml) were placed on top of the filter. After 3 h in 37°C, 5% CO2, the cells were wiped off the filter with a cell harvester and the filter was washed with medium before centrifuging at 1500 r.p.m. for 10 min. The


filter was carefully removed and the fluorescence was measured with a Wallac 1429 Victor2 (Turku, Finland) (excitation 485 nm and emission 535 nm). The migration to medium without chemokine was set to 100%, and a migration above 120% was considered to be a positive response. Results

Expression of chemokine receptor mRNA in multiple myeloma cell lines Total RNA from the MM cell lines were screened for their expression of the chemokine receptors CXCR1–CXCR5, CCR1–CCR5, CCR7, CCR8 and CX3CR mRNA by RNAse protection assay. All the cell lines examined were positive for CXCR4 and CCR1, although the bands for some of them were faint (Figure 1). U-266 1970, U-266 1984, U-1958 and Karpas 707 also expressed mRNA for CCR2. In addition, L363 showed mRNA expression for CCR4 (Figure 1). The expression was confirmed by RT-PCR (data not shown). In our RT-PCR survey of chemokine mRNA expression we also detected CXCR3 which was not apparent in the RPA analysis (data not shown). In addition, CCR6 that is not included in the RPA analysis could be detected by RT-PCR in all MM cell lines, except for L-363 and LP-1 (data not shown).

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Since our data indicated that the cells expressed transcripts for CXCR3, CXCR4, CCR1, CCR2 and CCR6, the cell lines U266 1970, U-266 1984, U-1958, Karpas 707, L363 and LP-1 were analyzed further by flow cytometry to measure if these cell lines also expressed these chemokine receptors on their surface. All cell lines were found to express the chemokine receptors CXCR4, and CCR1, and to some degree CCR6 (Figure 2). All cell lines except for U-266 1984 also expressed CXCR3, while the expression of CCR2 was very weak or absent on the cell lines investigated.

Chemokine-induced calcium mobilization in MM cells Receptor expression alone has not proven to be an adequate parameter to predict functionality of the receptor. Since binding of chemokines to their receptors causes a characteristic increase in cytosolic calcium in most cells, we examined intracellular calcium flux in FURA-2AM labelled MM cells. The chemokines used in the study are listed in Table 1. U1958 and L-363 showed an increase in [Ca2+]i after addition of SDF-1␣, MIP-1␣ and RANTES, whereas only SDF-1␣ induced [Ca2+]i in LP-1 (Figure 3). In contrast, U-266 1970, U-266

Figure 2 Flow cytometry analysis of chemokine receptor surface expression on MM cell lines. Flow cytometry analysis of the expression of CCR1, CCR2, CCR6, CXCR3 and CXCR4 on MM-derived cell lines is shown. The filled histogram represents the isotype-matched control antibody. Leukemia


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Table 1

Chemokines used in this study and their receptors

Chemokine

Receptor

MCP-1 (CCL2) MIP-1␣ (CCL3) RANTES (CCL5) MIP-3␣ (CCL20) IP-10 (CXCL10) SDF-1 (CXCL12)

CCR2 CCR1 CCR1, CCR3, CCR5 CCR6 CXCR3 CXCR4

1984 and Karpas 707 did not mobilize Ca2+ after addition of any of these chemokines (Figure 3). No increase in [Ca2+]icould be observed in any of the MM cell lines after addition of MCP-1, IP-10 or MIP-3␣ (data not shown).

Chemotaxis of MM cells in response to chemokines To analyze further the functionality of the receptors expressed on MM cells we performed a chemotaxis assay in which MM cells were evaluated for their ability to migrate to chemokines at 10, 100, and 500 ng/ml. All MM cells lines, except for U266 1984, exhibited a migratory response to SDF-1 and RANTES (Figure 4). U-266 1984 were found to be unresponsive or showed only minor migratory reponse to the chemokines tested. Some of the MM cell lines also exhibited a chemotactic response to MIP-1␣, MCP-1, IP-10 and MIP-3␣ (Figure 4).

Primary MM cells migrate in response to SDF-1, MIP1␣ and RANTES We next investigated the expression and function of chemokine receptors on primary MM cells purified from bone marrow aspirates. Our results from MM-derived cell lines demonstrated that CXCR4 and CCR1 were the most prominently expressed receptors in these cells. We therefore investigated the expression of CXCR4 and CCR1 on primary MM cells by flow cytometry and the migratory response of such cells to SDF-1, MIP-1␣ and RANTES. In Figure 5, a histogram of the expression of CXCR4 and CCR1 on primary MM cells from one out of five patients analyzed, is shown. The percentage positive cells of CXCR4 and CCR1 varied greatly among the patients, 2–95% for CXCR4 and 1–36% for CCR1. Despite variation in expression levels, primary MM cell clones from all patients tested migrated in response to SDF-1, MIP-1␣ and RANTES (Table 2). Discussion In this study we investigated the expression of chemokine receptors on primary MM cells and MM-derived cell lines and detected induced responses that are characteristic for the activation by chemokines, ie calcium mobilization and cell migration. Several of the chemokine receptors previously implicated in B cell migration could be identified, particularly CXCR4. An unexpected high expression of CCR1 was determined on both primary MM cells and cell lines. In accordance with previous reports we found that expression may not necessarily mean functionality. Furthermore, although some of the chemokines induced MM cell migration they did not induce Leukemia

Figure 3 Calcium mobilization by chemokines in MM cell lines. LP-1, L363, U-1958, Karpas 707, U-266 1970 and U-266 1984 cells were treated with chemokines as indicated. (SDF-1␣, 500 ng/ml; RANTES, 500 ng/ml; MIP-1␣, 500 ng/ml).

calcium mobilization in all of the cell lines (Figures 3 and 4). An explanation for this could be that calcium flux and the regulation of chemotaxis are dissociated since it has been shown that calcium flux is neither necessary nor sufficient for chemotaxis.34 Chemokine receptors involved in the homing of lympho-


207

Figure 4 Migration of MM cell lines in response to chemokines. The migration of MM cells were measured in 96-well chemotaxis chambers at concentrations giving optimal migration (mean ± s.e.m., n = 3).

cytes are often expressed on a discrete cell population during a specific phase of its development.5 Over the last few years the expression of chemokine receptor during B cell development has been delineated6,22,35 and includes CXCR4, CXCR5, CCR6 and CCR7. In this study we determined a high expression of CCR1 on all MM cell lines tested, as well as on primary MM cells (Figures 1 and 5). In healthy individuals, CCR1 is mainly expressed on monocytes and only very weakly on B cells.22,36 Primary MM cells and 5/6 MM cell lines migrated in response to the CCR1 ligands RANTES and/or MIP-1␣. High levels of MIP-1␣ are produced in bone marrow samples from MM patients, but not from control individuals.37 Thus, the expression of MIP-1␣ in the bone marrow and the unique expression of CCR1 on MM cells, suggest that

these are involved in the homing and confining of MM cells within the bone marrow. CCR6 expression within the B cell compartment is restricted to a subset of peripheral mature B cells.6,14 In vitro differentiated plasma cells appear not to express CCR6.14 In the same study by Krzysiek et al,14 they also investigated the expression of CCR6 on six MM cell lines and reported it to be absent. This contrasts to some degree with our findings where we could see a small expression of CCR6 on some of the MM cell lines tested and migration towards MIP-3␣, the ligand for CCR6. It should be pointed out though that none of these positive cell lines were investigated by Krzysiek et al. CXCR3 is expressed on activated T cells, but is usually lacking, or expressed at low levels, in resting T cells, B cells, monLeukemia


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Table 2

Migration of primary MM cells in response to SDF-1, MIP-1␣ and RANTES

SDF-1

Patient I Patient II Patient III a

MIP-1␣

10 ng/ml

500 ng/ml

10 ng/ml

500 ng/ml

10 ng/ml

100 ng/ml

149 ± 5a 95 ± 5 83 ± 14

155 ± 21 139 ± 8 175 ± 8

192 ± 5 130 ± 2 201 ± 10

134 ± 14 94 ± 3 133 ± 14

240 ± 6 137 ± 21 242 ± 19

236 ± 11 130 ± 9 199 ± 6

Mean migration ± s.e.m. of one experiment done in triplicate. A percentage ⬎120% was considered to be a postive migratory response.

Figure 5 Expression of CXCR4 and CCR1 on primary MM cells. The expression of CXCR4 and CCR1 on primary MM cells from bone marrow aspirates from one out of five patients analyzed is shown. The filled histogram represents the isotype-matched control antibody.

ocytes and granulocytes.38–41 In contrast to the lack of CXCR3 on normal B cells, the malignant counterparts of B cells have been reported to express CXCR3, particularly B cells from patients with CLL.42,43 Similarly to these findings, MM cells express CXCR3 (Figure 2). However, only three of them (U1958, Karpas 707 and L363) migrated in response to IP-10. A majority of peripheral blood B cells expresses CXCR5 and it has been shown that this expression is of importance for B cell migration into lymphoid follicles.8 In contrast to a number of B cell tumors, including chronic lymphocytic leukemia (CLL),23,24 we could not detect any expression of CXCR5 mRNA or protein in MM cells. The difference in CXCR5 expression on B-CLL and MM cells is likely due to the fact that these cells represent different stages of differentiation within the B cell lineage. Although CXCR4 is present on nearly all B cells, strong chemotactic responsiveness to its ligand, SDF-1, has been reported to occur only in B cells with an immature B cell phenotype.7,20,44,45 The expression levels and affinities of CXCR4 receptors for SDF-1 do not correlate with chemotactic responsiveness.44 In contrast to what we describe in this study it has been reported recently that human MM cell lines do not respond to SDF-1.44 However, two of the cell lines, ARH-77 and IM-9, tested in that study are not true MM cell lines, but are EBV-positive lymphoblastoid cell lines.31 Furthermore, another cell line tested by Fedyk et al,44 U-266BL, is probably identical to U-266 1984 used in this study which was found to be unresponsive. Of the six MM cell lines tested in our study, U-266 1984 was the only cell line found to be unresponsive to SDF-1␣, or to any other chemokine tested. U-266 1984 is a variant of the originally established U-266 1970 cell line which has altered its genotype and phenotype and adapted to be IL-6 independent during long-term in vitro culLeukemia

RANTES

ture.25 Thus, it is likely that the U-266 1984 line in addition to being IL-6 independent also has undergone other phenotypical changes as a consequence of the genetic changes that have occurred during long-term passage in vitro and so has become unresponsive to chemokines. In line with our results on the expression of CXCR4 on primary MM cells and MM cell lines is a recent study by Du¨rig et al,23 where they described that primary MM cells do express CXCR4. However, in that study they did not investigate the migratory response of the cells to SDF-1. Since receptor expression per se does not predict a cell’s chemotactic potential, one has to conclude that functional measurement of chemotaxis is the only way to determine the migratory response of a cell to a given chemokine. In this study we could demonstrate that primary MM cells migrate in response to SDF-1. SDF-1 is constitutively produced by bone marrow stromal cells.17,18 Production of SDF-1 within the bone marrow elicits chemotaxis of lymphoid progenitor cells to migrate and to adhere firmly to stromal cells. The adhesion, in turn, stimulates proliferation and B cell maturation.19 The aberrant functional expression of CXCR4 on MM cells may thus be one function that is involved in retaining this malignant cell within the bone marrow. This study has demonstrated that MM cells express chemokine receptors, particularly CXCR4 and CCR1, that might be of importance for the unique localization of MM cells to the bone marrow. Furthermore, SDF-1 and MIP-1␣ may not only direct, but also confine MM cells within the bone marrow, similar to the demonstrated mechanism described for progenitor B cell retention in the bone marrow by SDF-1.19 Thus, interfering with chemokines and their receptors may prove to be useful as new treatment for multiple myeloma.

Acknowledgements We thank Drs Karl Åkerman and Jyrki Kukkonen for help with ¨ sterborg, Karolinska Hospital, [Ca2+]i-analysis and Dr A O Stockholm, for providing the MM tumour samples. This work was supported by grants from the Swedish Cancer Society, Hans von Kantzow’s Foundation, Ollie and Elof Ericsson’s foundation, Erik, Karin and Go¨sta Selander’s Foundation, and Go¨ran Gustafsson’s Foundation.

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