Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells in bone marrow (BM) and recurrent or persistent infections. Toll-like receptors (TLRs) are essential in the host defense against infections and today 10 human TLRs (TLR1-TLR10) and one TLR-homolog (RP105) have been characterized. B cells express several TLRs (mainly TLR1, 6, 7, 9, 10 and RP105) and TLR-initiated responses in B cells include proliferation, anti-apoptosis effect and plasma cell (PC) differentiation. The present study was designed to analyze the role of TLRs in MM. We show that frequent expressions of TLRs were detected in cell lines from MM patients (minimum six TLRs in each). In comparison, only few TLRs (mainly TLR1 and or RP105) were found expressed in PCs from BM of healthy donors. In addition, TLR-specific ligands induce increased proliferation and survival of the MM cell lines, partially due to an autocrine interleukin-6 production. Importantly, we demonstrate that also PC from MM patients proliferates in response to TLR-specific ligands. In conclusion, TLR-ligands may contribute to increased growth and survival of MM cells in MM patients.
Multiple myeloma (MM) is an incurable B-cell malignancy characterized by accumulation of malignant plasma cells (MM cells) in the bone marrow (BM).1, 2 Multiple myeloma cells are dependent on factors in the BM microenvironment for survival and proliferation.3, 4, 5 Several cytokines, including interleukin-6 (IL-6), tumor necrosis factor (TNF), insulin-like growth factor-1 (IGF-1), IL-10, IL-21 and IL-15, are known to contribute to proliferation of MM cells in vitro.6, 7 However, the exact factors important for perpetuation of the disease are not fully understood.
Patients suffering from MM have reduced immune responses which often result in recurrent or persistent infections.8, 9 Toll-like receptors (TLRs) are essential in the host defense against infection of microorganisms.10, 11 Today, 10 human TLRs (TLR1-TLR10) and one TLR-homolog called RP105 (CD180), recognizing distinct structural components of pathogens, have been identified. B cells express many of the TLRs (TLR1, 2, 6, 7, 9, 10, RP105 and possibly TLR4 and 8) which mediate proliferation, PC differentiation and antiapoptotic effects in B cells.12, 13, 14, 15 For example, the TLR9 ligand CpG oligodeoxynucleotide (ODN), increases proliferation, survival and differentiation, as well as secretion of IL-6, IL-10, immunoglobulins (Igs) and it enhances expression of several activation and costimulatory proteins in B cells.12, 13, 16, 17 Furthermore, R-848, a synthetic antiviral product, activates cells via TLR7 and/or TLR8 and increases antibody secretion and cytokine production in B cells.18, 19 TLR1, 2 and 6 recognize various microbial components such as lipopeptides, which may activate B cells.20 In addition, TLR4/MD-2 and RP105/MD-1 both mediate bacterial lipopolysaccaride (LPS) responses in B cells.11, 20 MM patients suffer from frequent infections and because pathogens induce polyclonal activation of B cells through TLRs the possibility exists that these receptors are of significance for MM cell survival and proliferation. Therefore, TLR expressions and responses of primary MM cells and MM cell lines were addressed in the present study.
Materials and methods
Human multiple myeloma cell lines and culture medium
The human MM cell line ANBL-6 was a gift from Dr Diane Jelinek, Mayo Clinic (Rochester, MN, USA) and OH-2 was established in our laboratory.21 The IL-6-independent human MM cell lines RPMI-8266, NCI-H929, SKO-007, SKO-007(J3) and U266 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and OPM-2 and LP-1 were from the German Collection of Microorganisms, DSM, Heidelberg, Germany. All cell lines were cultured in RPMI 1640 (Life Technologies, Paisley, UK) containing 2 mM L-glutamine and 40 μg/ml gentamycin (referred to as medium) and in 5% CO2 at 37°C. ANBL-6 and OH-2 was maintained in medium supplemented with 10% heat inactivated fetal calf serum (FCS, Life Technologies, Paisley, UK) and 10% human serum (The Blood Bank, St Olav's Hospital, Trondheim, Norway), respectively, and IL-6 (1 ng/ml) (BioSource, Camarillo, CA, USA). U266 and RPMI-8266 were cultured in medium with 15% FCS. NCI-H929, SKO-007, SKO-007(J3) were cultured in medium with 10% FCS.
Toll-like receptor expression in multiple myeloma cell lines
RNA was isolated from ANBL-6 cells and OH-2 cells by RNAeasy mini kit (Qiagen, Crawley, UK). cDNA was synthesized from RNA (2 μg) at 37°C using DNA polymerase SuperScript™ II reverse transcriptase (RT) (Invitrogen, Oslo, Norway). Polymerase chain reaction (PCR) on cDNA (30 ng) was performed with 1 U of AmpliTaq Gold (Applied Biosystems, CA, USA). The primers for TLR7, TLR9, RP105 and MD-1 have previously been described.22, 23 The primers for TLR1-TLR6, TLR8, MD-2 and β-actin are shown in Supplementary Table 1. All primers were from Eurogentec, Seraing, Belgium. The PCR conditions were: 1 min at 95°C, 2 min at a primer-specific annealing temperature and 1 min at 72°C with a total of 40 cycles. Detection of TLR expression in the other seven MM cell lines were performed as described previously using the TLR primers shown in Supplementary Table 1.24 β-Actin RT-PCR was used as a control. Polymerase chain reaction products were applied on an agarose gel and visualized by ethidium bromide staining. Expressions of TLRs were defined as positive when a specific PCR band appeared in the sample with RT and not in the control sample without RT.
Toll-like receptor expression in bone marrow plasma cells from healthy donors
BM samples were obtained from 12 healthy donors after informed consent and the study was approved by the Ethical Committee of Copenhagen County Denmark in accordance with the Helsinki Declaration. Isolation of BM mononuclear cells and RNA were performed as previously described.25 Plasma cells and plasmablasts from healthy donors were sorted as CD38++/CD19+/CD45intermediate(i)/CD56− cells (purity above 96%) directly to PCR tubes by using a FACS Vantage (Becton Dickinson Immunocytometry Systems, CA, USA) in counter mode. Detection of TLR expressions were performed as described previously using the primers shown in Supplementary Table 1.24
Isolation of CD138+ plasma cells from multiple myeloma patients for Toll-like receptor-specific stimulation analysis
CD138+ PCs were isolated from six MM patients (classified according to Durie et al.26 and the International staging system (ISS)27) admitted to the Section of Hematology, St Olav's Hospital, Trondheim, Norway. Informed consent from the patients and approval from the Regional Ethical Committee were obtained. Patient characteristics are given in Supplementary Table 2. Lymphocytes from BM aspirates (patients MM1, MM2, MM5 and MM6) or peripheral blood (MM3 and MM4) were isolated by use of Isopaque–Ficoll separation medium (Lymphoprep; Nycomed, Oslo, Norway) and PCs were sorted by immunomagnetic separation using Macs CD138 MicroBeads (Miltenyi Biotec, CA, USA) according to the manufacturer's instructions. Cells were analyzed using a mouse monoclonal antibody specific for human CD138 (FITC, Serotec, Oxford, UK) and a FACscan flow cytometer (Becton Dickinson, CA, USA) demonstrating more than 95% pure CD138+ PC.
A synthetic lipopeptide Pam3Cys-Ser-Lys4 (Pam3Cys) from EMC Microcollections (Tübingen, Germany) was used to stimulate TLR1 and TLR2. Double-stranded polyribonucleotide poly (I-C) (Amersham Biosciences) was used as a TLR3-specific ligand.28 We used LPS (TLR 4 ligand) derived from Escherichia coli strain 0111:B4 purchased from Sigma and re-extracted by phenol chloroform.29 The ligand for TLR5, flagellin, was a kind gift from Dr Kelly D Smith (ISB, Seattle, USA).30 Synthetic 2-kDa macrophage-activating lipoprotein (MALP-2) derived from the structure of Mycoplasma fermentans MALP-2 (EMC Microcollections, Tübingen, Germany) was used as a ligand for TLR2 and TLR6. R-848 (resiquimod) from GLSynthesis (Worcester, MA, USA) was used as a ligand for TLR7 and/or TLR8.19 The TLR9 ligand, phosphorothioated ODN 5-′IndexTermTCGTCGTTTTGTCGTTTTGTCGTT-3′ (CpG 2006) was purchased from TIB MOLBIOL Syntheselabor (Berlin, Germany). Titration studies for each TLR ligand were performed on MM cell lines (OH-2 and/or ANBL-6) to determine the optimal concentration. The following concentrations of TLR ligands were used: Pam3Cys (1 μg/ml), poly (I-C) (50 μg/ml), LPS (200 ng/ml), Flagellin (1 μg/ml), MALP-2 (1 μg/ml), R-848 (1 μg/ml)19 and CpG 2006 (3 μg/ml).13 Neutralizing monoclonal anti-human IL-6 antibody (R&D Systems, Abingdon, UK) and an isotype control (mouse IgG1) were used to analyze the role of IL-6 following TLR stimulation. Amounts of IL-6 in culture medium were measured with human IL-6 ELISA (R&D Systems, Abingdon, UK). Samples were stored at −20°C until analysis.
Cells were seeded in 96-well (∼5 × 104 cells per well in 200 μl medium) plates (Corning Costar, Corning, NY, USA). The medium was supplemented with 10% FCS (10% human A+ serum for OH-2 cells) and cytokines, blocking antibodies and/or TLR-specific ligands in triplicate as indicated. After 54 h, cells were pulsed with 1 μCi 3H-thymidine (NEN Life Science Products, Boston, MA, USA) per well and were harvested 18 h later with a Micromate 96 cell harvester (Packard, Meriden, CT, USA) and beta radiation was measured with a Matrix 96 beta counter (Packard). For long-time culture experiments, 1 × 106 cells were cultured in 5 ml medium supplemented with 10% FCS or human A+ serum and IL-6 or TLR ligands as indicated. The cells were counted and media with supplements were replenished and the cell concentration were adjusted to 2 × 105 cells per ml every fourth day.
Cells were cultured for 72 h, with cytokines and TLR ligands as indicated, before viability and apoptosis were evaluated by annexin-V and propidium iodide (PI) binding (APOPTEST- FITC kit, Nexins Research, Hoeven, Netherlands) determined using a FACscan flow cytometer (Becton Dickinson, CA, USA).
Statistical significances of differences were determined by one-way ANOVA with Bonferroni's multiple comparison tests. A P-value of <0.05 was considered significant.
Human multiple myeloma cell lines express several types of Toll-like receptors and Toll-like receptor-specific ligands induce proliferation and increased survival
We analyzed mRNA expression of TLRs in nine MM cell lines (Table 1, upper panel and Figure 1a). High frequencies of TLRs were found in these cell lines as each expressed minimum six different TLR molecules. In comparison, by analysing BMPCs from 12 healthy donors we found that six of them did not express any TLRs, whereas the rest expressed mainly TLR1 and/or RP105 (Table 1, lower panel). To elucidate if the TLRs expressed in MM cell lines were functional, we incubated four MM cell lines with various TLR ligands and measured cell proliferation. As shown in Figure 1b, Pam3Cys (ligand for TLR1 and 2) and flagellin (TLR5), were more potent inducers of proliferation in OH-2 cells than IL-6. Significantly increased proliferation of OH-2 cells were also induced by MALP-2 (TLR2 and 6) and R-848 (TLR7). Pam3Cys-Ser-Lys4 and LPS (TLR4) were found to be as effective as IL-6 to induce proliferation of ANBL-6 cells (Figure 1c). In contrast, poly (I-C) (TLR3), flagellin and CpG ODN (TLR9) seemed to have a negative effect on the proliferation of ANBL-6 cells. Significantly increased proliferation was induced in the IL-6-independent cell line (RPMI-8226) in response to LPS and MALP-2 (Supplementary Figure 1). Although expressing several TLRs, no increase in proliferation was observed in U266 cells upon TLR-specific stimulations (data not shown).
In order to see if the TLR ligands were able to replace IL-6 in a long-time culture of IL-6-dependent MM cell lines, we cultured OH-2 cells and ANBL-6 in the presence of selected TLR ligands for 24 and 28, days respectively (Figure 1d and e). We demonstrate that the amount of OH-2 cells obtained after culture for 24 days in Pam3Cys was similar to that obtained by IL-6. Similarly, LPS was able to replace IL-6 in a long-time culture of ANBL-6 cells, while Pam3Cys could partially replace IL-6 in culture medium. Finally, we show that flagellin, MALP-2, R-848 and CpG ODN have a positive effect on the viability of OH-2 cells (Figure 1f and Supplementary Figure 2a) and LPS on the viability of ANBL-6 cells (Figure 1g and Supplementary Figure 2b). All together, these data demonstrate that TLR ligands are able to induce proliferation and increased survival in MM cell lines.
Toll-like receptor-specific ligands induce proliferation of CD138+ plasma cells from multiple myeloma patients
We next investigated if TLR stimulation affected the proliferation of CD138+ PCs from MM patients. Plasma cells were isolated from six MM patients (MM1-MM6) and the proliferation was determined after stimulation with TLR-specific ligands (Figures 2a and 3d). Several TLR ligands induced increased proliferation of the patient cells, with R-848 being the most frequent inducer of proliferation (five of six patients). Importantly, some of the TLR ligands were equally effective as IL-6 to induce proliferation. This was true, particularly for PCs from patient MM5 (Figure 2a), where Pam3Cys, LPS, MALP-2 and R-848 induced proliferation in the same range as seen by IL-6. We were also able to detect a small increase in viability of PC from patient MM3 (Figure 2b and Supplementary Figure 2c) following stimulation with TLR ligands. As observed in the MM cell lines variable responses to CpG ODN and poly (I-C) were found in the patients. CpG ODN had a negative effect on the proliferation in three MM patients (MM1, MM2 and MM6) and poly (I-C) reduced the proliferation in three patients (MM1, MM5 and MM6). In addition, poly (I-C) demonstrates a negative effect on the viability of cells from patient MM3. Overall, TLR-specific ligands are potent inducers of proliferation in PCs from MM patients, but, variations exist between patients in their response to the TLR ligands.
Toll-like receptor-specific ligands induce proliferation partly due to autocrine interleukin-6 secretion
The fact that TLR ligands are effective inducers of proinflammatory cytokines, including IL-6, prompted us to investigate a possible autocrine role of IL-6 in the proliferation of PCs and cell lines from MM patients. Pam3Cys-Ser-Lys4 was the most potent inducer of IL-6 production (161 pg/ml) in OH-2 cells, while flagellin induced a small amount of IL-6 (∼10 pg/ml) (Figure 3a). Neutralizing IL-6 antibody partially inhibited TLR-induced proliferation of OH-2 cells (Figure 3b). In contrast, we were not able to detect IL-6 following stimulation of ANBL-6 cells with TLR ligands (four different experiments, data not shown). However, the proliferation induced by Pam3Cys and LPS was completely inhibited in the presence of the neutralizing antibody to IL-6, suggesting that the amount of IL-6 released from ANBL-6 cells is below level of detection, but enough to induce proliferation (Figure 3c). Similarly, the increase in proliferation of PCs from a MM patient (MM6) induced by LPS and R-848 was completely inhibited by a neutralizing IL-6 antibody (Figure 3d), although no release of IL-6 was detected (data not shown). Finally, R-848 was the strongest stimulator of IL-6 production in three of four MM patients analyzed (81 pg/ml in MM1, 194 pg/ml in MM2 and 886 pg/ml in MM3, Supplementary Figure 3). In conclusion, the increased proliferation in cell lines and PCs from MM patients following TLR stimulation is partially due to autocrine IL-6 production.
In this paper, we show that TLR ligands induce proliferation of PCs from MM patients and MM cell lines. The frequent TLR expression in MM cell lines observed here, suggests a role of TLR specific ligands in the MM pathogenesis. The number of TLRs expressed in B cells increases during differentiation.13 However, it is not known whether TLRs are expressed in the last stage of B-cell differentiation, that is in the plasmablasts and PCs. We revealed an almost absence of TLRs in BMPCs and plasmablasts from 12 healthy donors, and if present it was mainly TLR1 and or RP105. Thus, our data suggest that the frequency of TLR expression described in memory B cells13 normally decreases when they differentiate into PCs. It has been demonstrated that cytokines such as INFγ, IL-4 and IL-6 increase expression of TLRs.31, 32 Toll-like receptors can also be upregulated by stimulation of the TLRs itself, by engagement of the B-cell receptor and following heat shock.13, 17, 32, 33 Increased level of IL-6 in BM of MM patients is common, thus it is tempting to speculate that IL-6 and possibly other factors in the BM microenvironment of MM patients are involved in the frequent TLR expression found in MM cells. Alternatively, TLR expression may also be induced during culture of these MM cell lines. However, in some of the MM patient samples several TLR ligands (Pam3Cys, LPS, Flagellin, MALP-2 and R-848) significantly increased cell proliferation, supporting a frequent and functional expression of TLRs in MM cells. In OH-2 cells, Pam3Cys and flagellin were found to be stronger inducers of proliferation than IL-6, which is considered among the most potent growth and antiapoptotic factors for MM cells, both in vitro and in vivo.7, 34 Moreover, Pam3Cys could replace IL-6 in long-time cultures of the IL-6-dependent cell lines OH-2 and partially for ANBL-6. In addition, LPS was found to induce proliferation and to be a substitute for IL-6 in a long-time culture of ANBL-6 cells. Bone marrow stromal cells are recognized as the most important producer of IL-6 in the BM, however it has been reported that some MM cells secrete IL-621, 35 and grow in an autocrine fashion.7 We detected production of IL-6 in PCs from MM patients as well as in OH-2 cells following stimulation with several of the TLR-specific ligands. The autocrine role of IL-6 was further supported by neutralizing IL-6 antibody partially inhibiting the TLR-induced proliferation. However, LPS and MALP-2 induced an increased proliferation of the IL-6-independent MM cell line, RPMI-8226, indicating other mechanisms than autocrine IL-6 may also be involved. It is not clear whether normal human B cells are able to recognize LPS. Expression of TLR4 has been reported, while others conclude that human B cells neither express TLR4 nor respond to LPS.14, 31, 36 Recent data demonstrate that RP105 and its coreceptor, MD-1, inhibit the LPS response in monocytes, while others have shown that they augment the LPS response in B cells.37, 38 RP105 and MD-1 mRNA were detected in the two cell lines responding well to LPS. CpG ODN, which is a strong stimulator of proliferation in B cells,16 induced only a small increase in proliferation of OH-2 and RPMI-8226 cells, and in one patient but reduced the proliferation of PCs from three patients and of ANBL-6. However, CpG did not reduce the survival of ANBL-6 cells. Opposing responses were also found for the TLR3 ligand, poly (I-C), in MM patients and in the cell lines. The reason for some TLR ligands having different effects is not known. However, both induction of proliferation and apoptosis has been observed in B-cell lymphomas stimulated with CpG ODN.39, 40 Taken together, our data show that several TLR ligands increase survival and proliferation of PCs and cell lines from MM patients.
The increased susceptibility for infections observed in MM patients support the pathological significance of TLR expression in MM.8, 9 TT-virus have been detected in the BM of MM patients, however, data on a possible role of bacterial or viral infections in MM is limited.41 Proliferation stimulated via TLRs may provide a link between inflammation and progression of MM. Possible endogenous TLR-specific ligands includes proteins released from apoptotic or necrotic cells, heat shock proteins, heparan sulfate and DNA, but their role as TLR ligands is controversial.42, 43, 44, 45 Nevertheless, several endogenous ligands are present in the BM of MM patients and may represent a possible source of TLR ligands. In conclusion, TLR-ligands may contribute to increased growth and survival of MM cells in MM patients, partially due to an autocrine IL-6 production. The involvement of TLRs in the pathogenesis of MM represents a possible new aspect of progression and maintenance of MM.
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We thank Marianne Lodahl at Herlev Hospital, University of Copenhagen, Denmark for technical assistance and Dr med. Anders Waage at St Olavs Hospital, Trondheim University Hospital, Norway for providing some of the patient material. This work was supported by grants from the Central Norway Regional Health Authority, Norwegian University of Science and Technology, the Norwegian Cancer Society and the Norwegian Research Council.
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Bohnhorst, J., Rasmussen, T., Moen, S. et al. Toll-like receptors mediate proliferation and survival of multiple myeloma cells. Leukemia 20, 1138–1144 (2006). https://doi.org/10.1038/sj.leu.2404225
- Toll-like rfeceptor
- multiple myeloma
- plasma cells
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