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 receptors in human multiple myeloma CMo¨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 tumor characterized by its , and to their pattern of expression of adhesion selective localization in the . The mechanisms that molecules.6 The chemokine receptors implicated in B cell contribute to the multiple myeloma cell recruitment to the bone marrow microenvironment are not well understood. Chemo- migration and proliferation include CXCR4, CXCR5, CCR2, 7–14 kines play a central role for trafficking and homing. CCR6 and CCR7. In this study we have investigated expression and functional The chemokine stromal cell-derived factor-1 (SDF-1) and its importance of chemokine receptors in MM-derived cell lines corresponding receptor CXCR4 have been shown to be essen- and primary MM cells. We found that MM cell lines express tial for bone marrow myelopoiesis and B lymphopoiesis.15,16 functional CCR1, CXCR3 and CXCR4 receptors, and some also SDF-1 is constitutively expressed at high levels by bone mar- CCR6. Although only a minority of the cell lines responded by 17,18 calcium mobilization after agonist stimulation, a migratory row stromal cells. CXCR4 appears to participate in the response to the CCR1 ligands RANTES and MIP-1␣ was regulation of B lymphopoiesis by confining precursors within obtained in 5/6 and 4/6, respectively, of the cell lines tested. the bone marrow microenvironment.19 The chemotactic Five out of six cell lines showed a response to the CXCR4 responsiveness of B cells to SDF-1 appears to be related to ligand SDF-1. In addition, 3/6 cell lines migrated in response to their differentiation stages, where mature forms of B cells ␣ MIP-3 and IP-10, ligands for CCR6 and CXCR3, respectively. although still expressing CXCR4, do not migrate towards SDF- The expression of CXCR4 and CCR1 and the migration to their 7,20–22 ligands, SDF-1, and RANTES and MIP-1␣, respectively, were 1. Human MM cells have been demonstrated to express also demonstrated in primary MM cells. These findings suggest CXCR4, but the functionality of these receptors has not been that expression and the migratory capacity investigated previously.23 of MM cells to their ligands are relevant for the compartmental- Here, we systematically investigated the expression of ization of MM cells in the bone marrow. chemokine receptors in MM cell lines. The functionality of Leukemia (2003) 17, 203–210. doi:10.1038/sj.leu.2402717 receptors was tested by agonist-induced calcium mobilization Keywords: multiple myeloma; migration; CCR1; CXCR4; RANTES; MIP-1␣; SDF-1 and cell migration. We also investigated functional expression of CXCR4 and CCR1 in primary MM cells.

Introduction Materials and methods

Multiple myeloma (MM) is a monoclonal expansion of malig- Cells nant cells with a plasmablast–plasma cell morphology that is almost exclusively localized to the bone marrow. MM cells A panel of human MM cell lines was used for the study: U- appear to be derived from a post germinal centre B cell as 266 1970,24 U-266 1984,25 U-1958,26 Karpas 707,27 LP-1,28 they express somatically mutated Ig heavy chain .1 The L-363,29 HL407E and HL407L.30 All the cell lines are negative mechanisms of the selective homing of MM cells to the bone for EBV, express CD138, and are classified as true MM cell marrow compartment are poorly understood. It is possible that lines.31 The cells were cultured in RPMI-1640 supplemented the bone marrow localization reflects the expression of chem- with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml otactic receptors on MM cells that directs their migration to penicillin and 50 ␮g/ml streptomycin (Life Technologies, Ren- the bone marrow. frewshire, UK). U-266 1970, U-1958, and HL407E are IL-6- Chemokines are a superfamily of that play a criti- dependent cell lines and were grown on a layer of IL-6-pro- cal role in the selective recruitment and homing of leukocytes ducing human fibroblast lines AG1523 (The Human Mutant by acting as chemotaxins.2 The chemokines can be classified Genetic Cell Repository, Camden, NJ, USA). Medium was into four subfamilies, C, CC, CXC and C(X)3C, based on the replenished twice a week. 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 Purification of primary MM cells chemokines mediate their effects by binding to seven trans- membrane-spanning, G- coupled receptors. Bone marrow samples were obtained from patients with The role of chemotactic factors in directing migration of gra- newly diagnosed MM. The use of fresh human tissue was nulocytes, , and T has been exten- approved by the local ethical committee according to the Hel- sively investigated, but less is known about B lymphocyte sinki Declaration and MM BM cells were obtained after infor- chemoattractants and their receptors.4,5 B cells at distinct dif- med consent. Mononuclear cells were purified by centrifug- ferentiation stages are differentially localized in primary ation over Ficoll–Hypaque density gradient (Pharmacia, lymphoid tissues or bone marrow, probably due to their Uppsala, Sweden), washed and incubated with anti-CD138- expression of chemokine receptors, their responsiveness to coated microbeads according to the manufacturer’s instruc- tions (Miltenyi Biotec, Auburn, CA, USA). Positive selection of magnetically labelled cells using an LS column and Midi- Correspondence: G Nilsson, Laboratory of Tumor Biology, Depart- Ͼ ment of Genetics and Pathology, Rudbeck Laboratory, Uppsala Uni- MACS resulted in a purity of 98% plasma cells as judged versity, 751 85 Uppsala, Sweden; Fax: +46 18 558931 by morphological examination of MGG-stained cytospin Received 23 October 2001; accepted 1 July 2002 preparations. Chemokine receptors in multiple myeloma CMo¨ller et al 204 Analysis of RNA expression by RNase protection (PBMNC) was used as a positive control for both RPA and assay and RT-PCR RT-PCR.

Total RNA was prepared using the TriPure Isolation Reagent (Boehringer Mannheim, Mannheim, Germany). Expression of Flow cytometry analysis of chemokine receptor chemokine receptor mRNA was analyzed by RNase Protec- expression tion 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 The MM-derived cell lines Karpas 707, U-1958, U-266 1970, In addition to RPA analysis we also performed RT-PCR U-266 1984, LP-1 and L363, and primary MM cells were ana- analysis. The RNA was first reverse-transcribed with First lyzed by flow cytometry for cell surface expression of chemo- Strand cDNA Synthesis Kit for RT-PCR (Boehringer kine receptors as described.32 The mAbs used were: anti- Mannheim) according to the manufacturer’s protocol. PCR human CXCR4-PE, anti-human CCR2-PE, anti-human CCR6- Core Kit (Boehringer Mannheim) was used for the amplifi- PE, anti-human CXCR3-FITC (PharMingen), anti-human CCR1 cation reactions according to the manufacturer’s protocol. and anti-human CCR1-FITC (R&D Systems, MN, USA). When mRNA from a pool of resting and activated (Con A 3 ␮g/ml unlabelled mouse anti-human CCR1 mAb were used, a sec- ␮ ′ or LPS 20 g/ml) peripheral blood mononuclear cells ondary PE conjugated F (ab )2 fragment of rabbit anti-mouse immunoglobulins (DAKO, Glostrup, Denmark) was added.

Isotype-matched mouse IgG1 (DAKO), IgG2A (DAKO) and IgG2B (The Binding Site, Birmingham, UK) were used to detect non-specific background fluorescence.

Cytosolic calcium

2+ Changes in the intracellular calcium concentration ([Ca ]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’ bal- anced salt solution w/o phenol red (HBSS) (Life Technologies) supplemented with 1% FBS. The cells were loaded with Fura- 2 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 2+ magnetically stirred and thermostated quartz cuvette. [Ca ]i was measured using excitation at 340/380 nm in a dual-wave- length fluorescence spectrophotometer (Hitachi-F2000, Toyo, Japan) after addition of the chemokines SDF-1␣, IP-10, MIP- 3␣, MCP-1, MIP-1␣ and RANTES (10–1000 ng/ml) (PeproTech, London, UK). Digitonin (Calbiochem-Novabi- ochem, 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

Migration studies were performed using the disposable 96- well chamber (ChemoTx; Neuroprobe, Gaithers- burg, 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 resus- pended 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 Figure 1 Expression of chemokine receptor mRNA in MM cell cell suspension (2 × 106 cells/ml) were placed on top of the 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; filter. After 3 h in 37 C, 5% CO2, the cells were wiped off the 7, U-1958; 8, U-266 1984; 9, U-266 1970; 10, human control RNA; filter with a cell harvester and the filter was washed with 11, PBMNC. medium before centrifuging at 1500 r.p.m. for 10 min. The

Leukemia Chemokine receptors in multiple myeloma CMo¨ller et al 205 filter was carefully removed and the fluorescence was meas- Surface expression of chemokine receptors ured with a Wallac 1429 Victor2 (Turku, Finland) (excitation 485 nm and emission 535 nm). The migration to medium Since our data indicated that the cells expressed transcripts without chemokine was set to 100%, and a migration above for CXCR3, CXCR4, CCR1, CCR2 and CCR6, the cell lines U- 120% was considered to be a positive response. 266 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 Results surface. All cell lines were found to express the chemokine receptors CXCR4, and CCR1, and to some degree CCR6 Expression of chemokine receptor mRNA in multiple (Figure 2). All cell lines except for U-266 1984 also expressed myeloma cell lines CXCR3, while the expression of CCR2 was very weak or absent on the cell lines investigated. 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 pro- Chemokine-induced calcium mobilization in MM tection assay. All the cell lines examined were positive for cells CXCR4 and CCR1, although the bands for some of them were faint (Figure 1). U-266 1970, U-266 1984, U-1958 and Karpas Receptor expression alone has not proven to be an adequate 707 also expressed mRNA for CCR2. In addition, L363 parameter to predict functionality of the receptor. Since bind- showed mRNA expression for CCR4 (Figure 1). The expression ing of chemokines to their receptors causes a characteristic was confirmed by RT-PCR (data not shown). In our RT-PCR increase in cytosolic calcium in most cells, we examined survey of chemokine mRNA expression we also detected intracellular calcium flux in FURA-2AM labelled MM cells. CXCR3 which was not apparent in the RPA analysis (data not The chemokines used in the study are listed in Table 1. U- 2+ shown). In addition, CCR6 that is not included in the RPA 1958 and L-363 showed an increase in [Ca ]i after addition of analysis could be detected by RT-PCR in all MM cell lines, SDF-1␣, MIP-1␣ and RANTES, whereas only SDF-1␣ induced 2+ except for L-363 and LP-1 (data not shown). [Ca ]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 anti- body.

Leukemia Chemokine receptors in multiple myeloma CMo¨ller et al 206 Table 1 Chemokines used in this study and their receptors

Chemokine Receptor

MCP-1 (CCL2) CCR2 MIP-1␣ (CCL3) CCR1 RANTES (CCL5) CCR1, CCR3, CCR5 MIP-3␣ (CCL20) CCR6 IP-10 (CXCL10) CXCR3 SDF-1 (CXCL12) CXCR4

1984 and Karpas 707 did not mobilize Ca2+ after addition of any of these chemokines (Figure 3). No increase in 2+ [Ca ]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 U- 266 1984, exhibited a migratory response to SDF-1 and RANTES (Figure 4). U-266 1984 were found to be unrespon- sive or showed only minor migratory reponse to the chemo- kines tested. Some of the MM cell lines also exhibited a chem- otactic response to MIP-1␣, MCP-1, IP-10 and MIP-3␣ (Figure 4).

Primary MM cells migrate in response to SDF-1, MIP- 1␣ and RANTES

We next investigated the expression and function of chemok- ine receptors on primary MM cells purified from bone marrow aspirates. Our results from MM-derived cell lines demon- strated 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 Figure 3 Calcium mobilization by chemokines in MM cell lines. In this study we investigated the expression of chemokine LP-1, L363, U-1958, Karpas 707, U-266 1970 and U-266 1984 cells receptors on primary MM cells and MM-derived cell lines and were treated with chemokines as indicated. (SDF-1␣, 500 ng/ml; detected induced responses that are characteristic for the acti- RANTES, 500 ng/ml; MIP-1␣, 500 ng/ml). vation by chemokines, ie calcium mobilization and cell migration. Several of the chemokine receptors previously implicated in B cell migration could be identified, particularly calcium mobilization in all of the cell lines (Figures 3 and 4). CXCR4. An unexpected high expression of CCR1 was determ- An explanation for this could be that calcium flux and the ined on both primary MM cells and cell lines. In accordance regulation of chemotaxis are dissociated since it has been with previous reports we found that expression may not neces- shown that calcium flux is neither necessary nor sufficient sarily mean functionality. Furthermore, although some of the for chemotaxis.34 chemokines induced MM cell migration they did not induce Chemokine receptors involved in the homing of lympho-

Leukemia Chemokine receptors in multiple myeloma CMo¨ller et al 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 these are involved in the homing and confining of MM cells a specific phase of its development.5 Over the last few years within the bone marrow. the expression of chemokine receptor during B cell develop- CCR6 expression within the B cell compartment is restricted ment has been delineated6,22,35 and includes CXCR4, CXCR5, to a subset of peripheral mature B cells.6,14 In vitro differen- CCR6 and CCR7. In this study we determined a high tiated plasma cells appear not to express CCR6.14 In the same expression of CCR1 on all MM cell lines tested, as well as on study by Krzysiek et al,14 they also investigated the expression primary MM cells (Figures 1 and 5). In healthy individuals, of CCR6 on six MM cell lines and reported it to be absent. CCR1 is mainly expressed on and only very This contrasts to some degree with our findings where we weakly on B cells.22,36 Primary MM cells and 5/6 MM cell could see a small expression of CCR6 on some of the MM lines migrated in response to the CCR1 ligands RANTES cell lines tested and migration towards MIP-3␣, the ligand for and/or MIP-1␣. High levels of MIP-1␣ are produced in bone CCR6. It should be pointed out though that none of these posi- marrow samples from MM patients, but not from control indi- tive cell lines were investigated by Krzysiek et al. viduals.37 Thus, the expression of MIP-1␣ in the bone marrow CXCR3 is expressed on activated T cells, but is usually lack- and the unique expression of CCR1 on MM cells, suggest that ing, or expressed at low levels, in resting T cells, B cells, mon-

Leukemia Chemokine receptors in multiple myeloma CMo¨ller et al 208 Table 2 Migration of primary MM cells in response to SDF-1, MIP-1␣ and RANTES

SDF-1 MIP-1␣ RANTES

10 ng/ml 500 ng/ml 10 ng/ml 500 ng/ml 10 ng/ml 100 ng/ml

Patient I 149 ± 5a 155 ± 21 192 ± 5 134 ± 14 240 ± 6 236 ± 11 Patient II 95 ± 5 139 ± 8 130 ± 294± 3 137 ± 21 130 ± 9 Patient III 83 ± 14 175 ± 8 201 ± 10 133 ± 14 242 ± 19 199 ± 6

aMean migration ± s.e.m. of one experiment done in triplicate. A percentage Ͼ120% was considered to be a postive migratory response.

ture.25 Thus, it is likely that the U-266 1984 line in addition to being IL-6 independent also has undergone other pheno- typical 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 pri- mary 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 poten- tial, 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 Figure 5 Expression of CXCR4 and CCR1 on primary MM cells. demonstrate that primary MM cells migrate in response to The expression of CXCR4 and CCR1 on primary MM cells from bone SDF-1. marrow aspirates from one out of five patients analyzed is shown. The SDF-1 is constitutively produced by bone marrow stromal filled histogram represents the isotype-matched control . 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, stimu- ocytes and granulocytes.38–41 In contrast to the lack of CXCR3 lates proliferation and B cell maturation.19 The aberrant func- on normal B cells, the malignant counterparts of B cells have tional expression of CXCR4 on MM cells may thus be one been reported to express CXCR3, particularly B cells from function that is involved in retaining this malignant cell within patients with CLL.42,43 Similarly to these findings, MM cells the bone marrow. express CXCR3 (Figure 2). However, only three of them (U- This study has demonstrated that MM cells express chemok- 1958, Karpas 707 and L363) migrated in response to IP-10. ine receptors, particularly CXCR4 and CCR1, that might be of A majority of peripheral blood B cells expresses CXCR5 and importance for the unique localization of MM cells to the it has been shown that this expression is of importance for B bone marrow. Furthermore, SDF-1 and MIP-1␣ may not only cell migration into lymphoid follicles.8 In contrast to a number direct, but also confine MM cells within the bone marrow, of B cell tumors, including chronic lymphocytic leukemia similar to the demonstrated mechanism described for progeni- (CLL),23,24 we could not detect any expression of CXCR5 tor B cell retention in the bone marrow by SDF-1.19 Thus, mRNA or protein in MM cells. The difference in CXCR5 interfering with chemokines and their receptors may prove to expression on B-CLL and MM cells is likely due to the fact be useful as new treatment for multiple myeloma. 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 Acknowledgements reported to occur only in B cells with an immature B cell phenotype.7,20,44,45 The expression levels and affinities of We thank Drs Karl Åkerman and Jyrki Kukkonen for help with 2+ CXCR4 receptors for SDF-1 do not correlate with chemotactic [Ca ]i-analysis and Dr A O¨ sterborg, Karolinska Hospital, responsiveness.44 In contrast to what we describe in this study Stockholm, for providing the MM tumour samples. This work it has been reported recently that human MM cell lines do not was supported by grants from the Swedish Cancer Society, respond to SDF-1.44 However, two of the cell lines, ARH-77 Hans von Kantzow’s Foundation, Ollie and Elof Ericsson’s and IM-9, tested in that study are not true MM cell lines, but foundation, Erik, Karin and Go¨sta Selander’s Foundation, and are EBV-positive lymphoblastoid cell lines.31 Furthermore, Go¨ran Gustafsson’s Foundation. 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 References study, U-266 1984 was the only cell line found to be unre- sponsive to SDF-1␣, or to any other chemokine tested. U-266 1 Bakkus MH, Heirman C, Van Riet I, Van Camp B, Thielemans K. 1984 is a variant of the originally established U-266 1970 cell Evidence that multiple myeloma Ig heavy chain VDJ genes contain line which has altered its genotype and phenotype and somatic mutations but show no intraclonal variation. Blood 1992; adapted to be IL-6 independent during long-term in vitro cul- 80: 2326–2335.

Leukemia Chemokine receptors in multiple myeloma CMo¨ller et al 209 2 Baggiolini M. Chemokines and leukocyte traffic. Nature 1998; ini M, MacLennan IC, Acha-Orbea H. Changing responsiveness 392: 565–568. to chemokines allows medullary plasmablasts to leave lymph 3 Mackay CR. Chemokines: immunology’s high impact factors. Nat nodes. Eur J Immunol 2001; 31: 609–616. Immunol 2001; 2: 95–101. 23 Du¨rig J, Schmucker U, Duhrsen U. Differential expression of 4 Kim CH, Broxmeyer HE. Chemokines: signal lamps for trafficking chemokine receptors in B cell malignancies. Leukemia 2001; 15: of T and B cells for development and effector function. J Leukoc 752–756. Biol 1999; 65: 6–15. 24 Nilsson K, Bennich H, Johansson S, Ponten J. Established immuno- 5 Moser B, Loetscher P. Lymphocyte traffic control by chemokines. globulin producing myeloma (IgE) and lymphoblastoid (IgG) cell Nat Immunol 2001; 2: 123–128. line from an IgE myeloma patient. Clin Exp Immunol 1970; 7: 6 Bowman EP, Campbell JJ, Soler D, Dong Z, Manlongat N, Picarella 477–489. D, Hardy RR, Butcher EC. Developmental switches in chemokine 25 Hellman L, Josephson S, Jernberg H, Nilsson K, Pettersson U. response profiles during B cell differentiation and maturation. J Immunoglobulin synthesis in the human myeloma cell line U-266; Exp Med 2000; 191: 1303–1318. expression of two immunoglobulin heavy chain isotypes (epsilon 7 D’Apuzzo M, Rolink A, Loetscher M, Hoxie JA, Clark-Lewis I, Mel- and alpha) after long-term cultivation in vitro. Eur J Immunol 1988; chers F, Baggiolini M, Moser B. The chemokine SDF-1, stromal 18: 905–910. cell-derived factor 1, attracts early stage B cell precursors via the 26 Jernberg H, Nilsson K, Zech L, Lutz D, Nowotny H, Scheirer W. chemokine receptor CXCR4. Eur J Immunol 1997; 27: 1788–1793. Establishment and phenotypic characterization of three new 8 Forster R, Mattis AE, Kremmer E, Wolf E, Brem G, Lipp M. A puta- human myeloma celll lines (U-1957, U-1958, and U-1996). Blood tive chemokine receptor, BLR1, directs B cell migration to defined 1987; 69: 1605–1612. lymphoid organs and specific anatomic compartments of the 27 Karpas A, Fischer P, Swirsky D. Human myeloma cell line carrying . Cell 1996; 87: 1037–1047. a Philadelphia . Science 1982; 216: 997–999. 9 Legler DF, Loetscher M, Roos RS, ClarkLewis I, Baggiolini M, 28 Pegoraro L, Malavasi F, Belloni G, Massaia M, Boccadoro M, Sag- Moser B. B cell-attracting chemokine 1, a human CXC chemokine lio G, Guerrasio A, Benetton G, Lombardi L, Coda R, Avanzi G. expressed in lymphoid tissues, selectively attracts B lymphocytes The human myeloma cell lineLP-1: versatile model in which to via BLR1/CXCR5. J Exp Med 1998; 187: 655–660. study early plasma cell differentiation and c-myc activation. Blood 10 Frade JM, Mellado M, del Real G, Gutierrez-Ramos JC, Lind P, 1989; 73: 1020–1027. Martinez-A C. Characterization of the CCR2 chemokine receptor: 29 Diehl V, Schaadt M, Kirchner H, Hellriegel KP, Gudat F, Fonatsch functional CCR2 receptor expression in B cells. J Immunol 1997; C, Laskewitz E, Guggenheim R. Long-term cultivation of plasma 159: 5576–5584. cell leukemia cells and autologous lymphoblasts (LCL) in vitro:a 11 Baba M, Imai T, Nishimura M, Kakizaki M, Takagi S, Hieshima comparative study. Blut 1978; 36: 331–338. K, Nomiyama H, Yoshie O. Identification of CCR6, the specific 30 Scibienski RJ, Paglieroni T, Caggiano V, Lemongello D, Gumer- receptor for a novel lymphocyte-directed CC chemokine LARC. J lock PH, Mackenzie MR. Factors affecting the in vitro evolution Biol Chem 1997; 272: 14893–14898. of a myeloma cell line. Leukemia 1992; 6: 940–947. 12 Yoshida R, Imai T, Hieshima K, Kusuda J, Baba M, Kitaura M, 31 Jernberg-Wiklund H, Nilsson K. Plasma cell and myeloma cell Nishimura M, Kakizaki M, Nomiyama H, Yoshie O. Molecular lines. In: Masters JRW, Palsson B (eds). Human cell culture, 3. cloning of a novel human CC chemokine EBI1-ligand chemokine Kluwer Academic Publishers: Dordrecht, 2000, pp 81–155. that is a specific functional ligand for EBI1, CCR7. J Biol Chem 32 Juremalm M, Hjertson M, Olsson N, Harvima I, Nilsson K, Nilsson 1997; 272: 13803–13809. G. The chemokine receptor CXCR4 is expressed within the mast 13 Nagira M, Imai T, Yoshida R, Takagi S, Iwasaki M, Baba M, Tabira cell lineage and its ligand SDF-1␣ acts as a mast cell chemotaxin. Y, Akagi J, Nomiyama H, Yoshie O. A lymphocyte-specific CC Eur J Immunol 2000; 30: 3614–3622. chemokine, secondary lymphoid tissue chemokine (SLC), is a 33 Frevert CW, Wong VA, Goodman RB, Goodwin R, Martin TR. highly efficient chemoattractant for B cells and activated T cells. Rapid fluorescence-based measurement of migration in Eur J Immunol 1998; 28: 16–23. vitro. J Immunol Methods 1998; 213: 41–52. 14 Krzysiek R, Lefevre EA, Bernard J, Foussat A, Galanaud P, Louache 34 Rabin RL, Park MK, Liao F, Swofford R, Stephany D, Farber JM. F, Richard Y. Regulation of CCR6 chemokine receptor expression Chemokine receptor responses on T cells are achieved through and responsiveness to inflammatory protein- regulation of both receptor expression and signaling. J Immunol 3alpha/CCL20 in human B cells. Blood 2000; 96: 2338–2345. 1999; 162: 3840–3850. 15 Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, 35 Hargreaves DC, Hyman PL, Lu TT, Ngo VN, Bidgol A, Suzuki G, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T. Defects of B-cell Zou YR, Littman DR, Cyster JG. A coordinated change in chemok- lymphopoiesis and bone-marrow myelopoiesis in mice lacking the ine responsiveness guides plasma cell movements. J Exp Med CXC chemokine PBSF/SDF-1. Nature 1996; 382: 635–638. 2001; 194: 45–56. 16 Ma Q, Jones D, Borghesan PR, Segal RA, Nagasawa T, Kishimoto 36 Su SB, Mukaida N, Wang J, Nomura H, Matsushima K. Preparation T, Bronson R, Springer TA. Impaired B-lymphopoiesis, myelopo- of specific polyclonal antibodies to a C-C chemokine receptor, iesis, and derailed cerebellar neuron migration in CXCR4-and CCR1, and determination of CCR1 expression on various types of SDF-1-deficient mice. Proc Natl Acad Sci USA 1998; 95: 9448– leukocytes. J Leukoc Biol 1996; 60: 658–666. 9453. 37 Choi SJ, Cruz JC, Craig F, Chung H, Devlin RD, Roodman GD, 17 Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T. Alsina M. Macrophage inflammatory protein 1-alpha is a potential Signal sequence trap: a cloning strategy for secreted and osteoclast stimulatory factor in multiple myeloma. Blood 2000; type I membrane proteins. Science 1993; 261: 600–603. 96: 671–675. 18 Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA. A 38 Qin SX, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher highly efficacious lymphocyte chemoattractant, stromal cell- M, Koch AE, Moser B, Mackay CR. The chemokine receptors derived factor 1 (SDF-1). J Exp Med 1996; 184: 1101–1109. CXCR3 and CCR5 mark subsets of T cells associated with certain 19 Ma Q, Jones D, Springer TA. The chemokine receptor CXCR4 is inflammatory reactions. J Clin Invest 1998; 101: 746–754. required for the retention of B lineage and granulocytic precursors 39 Piali L, Weber C, LaRosa G, Mackay CR, Springer TA, Clark-Lewis within the bone marrow microenvironment. Immunity 1999; 10: I, Moser B. The chemokine receptor CXCR3 mediates rapid and 463–471. shear-resistant adhesion-induction of effector T lymphocytes by 20 Bleul CC, Schultze JL, Springer TA. B lymphocyte chemotaxis the chemokines IP10 and Mig. Eur J Immunol 1998; 28: 961–972. regulated in association with microanatomic localization, differen- 40 Loetscher M, Loetscher P, Brass N, Meese E, Moser B. Lympho- tiation state, and B cell receptor engagment. J Exp Med 1998; 187: cyte-specific chemokine receptor CXCR3: regulation, chemokine 753–762. binding and localization. Eur J Immunol 1998; 28: 3696– 21 Honczarenko M, Douglas RS, Mathias C, Lee B, Ratajczak MZ, 3705. Silberstein LE. SDF-1 responsiveness does not correlate with 41 Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Lymphocyte- CXCR4 expression levels of developing human bone marrow B specific chemokine receptor CXCR3: regulation, chemokine bind- cells. Blood 1999; 94: 2990–2998. ing and gene localization. J Exp Med 1998; 187: 875–883. 22 Wehrli N, Legler DF, Finke D, Toellner KM, Loetscher P, Baggiol- 42 Jones D, Benjamin RJ, Shahsafaei A, Dorfman DM. The chemok-

Leukemia Chemokine receptors in multiple myeloma CMo¨ller et al 210 ine receptor CXCR3 is expressed in a subset of B-cell lymphomas decreases responsiveness of human bone marrow B lineage cells and is a marker of B-cell chronic lymphocytic leukemia. Blood to stromal-derived factor 1 (SDF-1). J Leukoc Biol 1999; 66: 2000; 95: 627–632. 667–673. 43 Trentin L, Agostini C, Facco M, Piazza F, Perin A, Siviero M, Gurri- 45 Vicente-Manzanares M, Montoya MC, Mellado M, Frade JM, del eri C, Galvan S, Adami F, Zambello R, Semenzato G. The chemok- Pozo MA, Nieto M, de Landazuri MO, Martinez-A C, Sanchez- ine receptor CXCR3 is expressed on malignant B cells and Madrid F. The chemokine SDF-1alpha triggers a chemotactic mediates chemotaxis. J Clin Invest 1999; 104: 115–121. response and induces cell polarization in human B lymphocytes. 44 Fedyk ER, Ryyan D, Ritterman I, Springer TA. Maturation Eur J Immunol 1998; 29: 2197–2207.

Leukemia