Expression and Function of Chemokine Receptors in Human Multiple Myeloma Cmo¨Ller, T Stro¨Mberg, M Juremalm, K Nilsson and G Nilsson
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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 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 B cell tumor characterized by its chemokines, and to their pattern of expression of adhesion selective localization in the bone marrow. 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 lymphocyte 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 chemokine receptor 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 genes.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 cytokines 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-protein 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, macrophages, and T lymphocytes 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 antibodies 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 chemotaxis 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.