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Keywords:

  • Multiple myeloma;
  • Toll-like receptor;
  • Toll-like receptor ligands;
  • interleukin-6;
  • nuclear factor-kappa B

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Toll like receptors (TLRs) are the major agents for innate immunity that recognize invading microbial products and regulate the growth of normal and malignant human B lymphocytes. Multiple myeloma (MM) is a clonal plasma cell malignancy, though the regulatory role of TLRs in MM plasma cells has been reported, the molecular mechanism remains unclear. We first compared the transcripts of TLR1 to TLR10 in MM patients and healthy donors and found that TLR2, -4 and -9 transcripts were higher in bone marrow mononuclear cells (BMMCs) from patients than those from donors; in addition the expression of TLR4 and TLR9 were higher in MM cells than normal cells as demonstrated by flow cytometric analyses. The ligands of these two TLRs were capable to promote the growth of MM cells and protect them from serum-deprivation-induced apoptosis but not normal plasma cells, which could be attenuated with anti-IL6 neutralizing antibodies or blockage of NF-κB activities. Further investigation demonstrated that these TLR ligands could trigger the nuclear translocation of NF-κB p65 and the activated NF-κB was sufficient to increase the expression of IL6 transcript in MM cells. These data suggested that activated NF-κB signalling probably plays a crucial role for the ligands of TLR4 and TLR9 to promote the growth and survival of MM cells partially through IL6 autocrine.

Multiple myeloma (MM) arises from an accumulation of malignant plasma B cells in the bone marrow (BM), which accounts for about 10% of all haematological malignancies (Kyle & Rajkumar, 2004; Kyle & Rajkumar, 2008). MM cells grow and survive depending on BM microenvironment and several cytokines, such as interleukin (IL) 6, IL10 and tumour necrosis factor α (TNF-α) (Anderson et al, 2002; Hideshima et al, 2003; Seidl et al, 2003). As normal plasma cells are inhibited and level of normal immunoglobulin (Ig) is low, MM patients are usually susceptible to infections caused by bacteria, viruses and fungi at diagnosis (Blade et al, 1996). However, the effects of these microbe infections on the growth of malignant plasma cells has not yet been fully revealed.

Toll like receptors (TLRs) play a pivotal role in sensing and initiating innate immune response (Iwasaki & Medzhitov, 2004; Pasare & Medzhitov, 2004; Akira et al, 2006). Ten human TLRs (TLR1-TLR10) have been identified, each of which recognizes a specific pathogen-associated molecular pattern (PAMP). Briefly, TLR1 and TLR2 are the receptors for lipopeptides (Manukyan et al, 2005), TLR3 is triggered by double-stranded RNA (dsRNA) (Alexopoulou et al, 2004), TLR4 recognizes lipopolysaccharide (LPS) (Poltorak et al,1998), TLR5 is the receptor for flagellin (Hayashi et al, 2001), TLR7 and TLR8 are triggered by single-stranded RNA (ssRNA) (Diebold et al, 2004; Heil et al, 2004) and TLR9 is activated by unmethylated CpG DNA (Hemmi et al, 2000). Upon the detection of pathogens, TLRs initiate a signalling cascade that leads to the activation of transcription factors, such as nuclear factor-kappa B (NF-κB) and interferon regulatory factors (IRFs), which eventually result in the production of pro-inflammatory cytokines and type-I interferons. The activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinases (JNK) upon stimulation of TLRs has also been reported (Akira, 2003; Kawai & Akira, 2006).

In present study, the comparative expression of TLR1 - TLR10 transcripts in bone marrow mononuclear cells (BMMCs) from MM patients, CD138 enriched MM patient cells and healthy donors were analysed with reverse transcription polymerase chain reaction (RT-PCR). The regulatory effect of TLR ligands on growth and survival of MM cells was studied; in addition a signalling cascade stimulated by the TLR ligands was elucidated.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Human bone marrow myeloma cells and myeloma cell lines

Bone marrow from 29 MM patients at diagnosis and 25 healthy donors were collected in this study with individual informed consent (Table I). Mononuclear cells were obtained by gradient centrifugation on Ficoll-hypaque medium (GE, Piscataway, NJ, USA). Primary myeloma cells from MM patients and normal plasma cells from healthy donors were purified using CD138 microbeads according to the instruction of the manufacturer (Miltenyi Biotech, Bergisch Gladbach, Germany). U266 (IL6 independent), RMPI-8226 (IL6 independent) and XG-7 (IL6 dependent) cell lines were kind gifts from Professor Xueguang Zhang, and were maintained in RPMI 1640 media supplemented with 15% fetal bovine serum, 2 mmol/l glutamine and 1% penicillin/streptomycin (Invitrogen/GIBCO-BRL, Carlsbad, CA, USA). XG-7 cells were maintained with additional 1 ng/ml recombinant IL6 (rIL6). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

Table I.   Clinical features of MM patients and healthy donors.
ParameterPatientsHealthy donors
  1. (–): No data.

  2. N/R, newly diagnosed/relapsed; BM, bone marrow; IDA, iron deficiency anaemia; CRF, chronic renal failure; Lung, lung infection.

Number (N/R)29 (27/2)25
Median age (range), years59 (33–78)48 (22–64)
Male/female16/1313/12
TypeIgG 8; IgA 2; IgG,κ 4; IgA,κ 2; IgD,λ 1; IgD,κ 1; λ 2; (–) 9Normal 19; IDA 6
Stage (Durie–Salmon)II A 5/20; II B 1/20; III A 6/20; III B 8/20 
Organs involvedLung 4/20; CRF 9/20; Anaemia 9/20; Osteolysis 9/20 
Average plasma cells in BM at diagnosis (%)36·1(7·5–62·5)1·1(0·5–2·0)

Chemicals, antibodies, plasmids and transfections

SP600125 (JNK inhibitor), PD169316 (p38 inhibitor) and PD98059 (ERK1/2 inhibitor) were purchased from Calbiochem (San Diego, CA, USA). Pam3Csk4 and LPS were purchased from InvivoGen (San Diego, CA, USA). TLR9 ligand, CpG oligonucleotide, 5-′TCGTCG TTTTGTCGTTTTGTCGTT-3′ (CpG 2006) was purchased from Sangon (Shanghai, China). Antibodies against tubulin and Poly-ADP ribose polymerase (PARP) were from BD Biosciences (San Jose, CA, USA); antibody against NF-κB p65 was from Cell Signaling Technology (Danvers, MA, USA); and peroxidase-conjugated secondary antibodies were from Santa Cruz (Santa Cruz, CA, USA). Monoclonal human IL6 neutralizing antibody was purchased from R&D Systems (Abingdon, UK). Phycoerythrin (PE)-conjugated antibodies against human TLR2, TLR4 and TLR9 were purchased from eBioscience (San Diego, CA, USA).

Plasmid RcCMVIκBα-SR (IκBα-SR) expressed a super-repressor form of IκBα, whose serines 32 and 36 were mutated to alanines (Traenckner et al, 1995). Plasmids pRc-β-actin-3HA-IKKα and –IKKβ (IKKα/β) were provided by Dr. M. Karin (University of California San Diego, CA, USA; DiDonato et al, 1997; Zandi et al, 1997). U266 cells and RPMI 8226 cells were transfected with liposome reagent (Fugene reagent; Roche, Indianapolis, IN, USA) mixed with plasmid DNA at a 5:1 ratio. All transfections were performed using equal total amounts of plasmid DNA normalized to its empty vector. The transfection efficiency was monitored by detecting the activities of β-galactosidase in the cell lysates (β-Galactosidase Enzyme Assay kitC; Promega, Madison, WI, USA) after co-transfection with the pSV-β-galactosidase (Promega) vector as the control.

RNA extraction and real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. In order to remove the genomic DNA, the total RNA was treated with DNase I (Takara Bio INC, Dalian, China), and then reversed transcribed using Superscript™ II Reverse Transcriptase (Invitrogen Life Technologies) and random hexamer primers. Real-time PCR was performed in the Bio-Rad Icycler IQ detection system (Bio-Rad Laboratories, Hercules, CA, USA) using the SYBR(R) Premix Ex Taq™ (Takara Bio INC) according to the manufacturer’s instructions. Briefly, the same amount of cDNA template was used for each PCR with 250 nmol/l forward and reverse primers (Table S1) (Holmlund et al, 2002; Nagase et al, 2003) in a total volume of 25 μl. After an initial denaturation step at 95°C for 5 min, 45 cycles of a three-step PCR with a single fluorescence measurement were undertaken (95°C for 15 s, 60°C for 30 s, and 72°C for 30 s). The PCR products were also subjected to melting curve analysis for verification of single amplicons and absence of primer dimmers (data not shown). Quantitative (Q)-RT-PCR and data analysis were performed on an iCycler iQ system, using iCycler iQ real-time detection software (Bio-Rad).

Flow cytometry

Freshly thawed BMMCs from patients and healthy donors were enumerated, and then 500 μl cells (2 × 106 cells/ml) were transferred into 5 ml Falcon tubes and incubated with phycoerythrin (PE)-conjugated antibodies against human TLR2, TLR4 and TLR9 respectively for 2 h, according to the manufacturer’s instructions, these cells were then fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS), and analysed by flow cytometry (Beckman Coulter, Brea, CA, USA). for each sample 104 cells were acquired. Intracellular staining of TLR9 required fixation and permeabilization with Cytofix/Cytoperm and washing with Perm/Wash (BD Biosciences) before staining. The mean fluorescence intensity (MFI) of the individual TLRs was determined using BD FCASDiva software (BD Bioscience).

Evaluation the expression of CD138 in BMMCs with flow cytometry

Bone marrow mononuclear cells from patients were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mmol/l glutamine, 1% penicillin/streptomycin (Invitrogen/GIBCO-BRL). An aliquot of cells (1 × 106 cells/ml) were plated in 24-well plates, and then LPS (200 ng/ml), Pam3Csk4 (1 μg/ml) and CpG DNA (1 μg/ml) were added respectively. After 72 h, cells were collected and stained with PE-conjugated anti-human CD138 antibody (BD Bioscience) according to the manufacturer’s instruction. Briefly, after the cells were fixed with 3% paraformaldehyde in PBS, they were analysed by flow cytometry (Beckman Coulter) and the data was analysed with BD FCASDiva software (BD Bioscience).

MTT assay

Multiple myeloma cell growth was monitored using the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)–based assay, according to the manufacturer’s protocol. Briefly, in a 96-well flat-bottom plate, 104 cells in 200 μl RPMI 1640 media were plated and then treated with vehicle, LPS (200 ng/ml), Pam3Csk4 (1 μg/ml) and CpG DNA (1 μg/ml) respectively, for 24, 48 and 72 h. After treatment, 20 μl of 5 mg/ml MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 4 h at 37°C. The plate was then spun at 1000 g for 5 min and the media were removed. The remaining cells were solubilized with 100 μl dimethyl sulfoxide (Sigma-Aldrich) and incubated for 10 min at room temperature. Plates were then analysed with a microplate reader (Bio-Rad) for the absorbance at 490 nm. The net absorbance was determined by comparing the absorbance reading of each set of test wells to a set of control wells in which vehicle were added. The following equation was used to determine the growth of the cells treated with various drugs compared to that treated with vehicle, ([absorbance of vehicle – absorbance of test]/absorbance of vehicle) × 100%.

Apoptosis assay

About 1 × 106 CD138 purified cells from two MM patients, RMPI-8226, XG-7 cell lines and normal plasma cells were cultured in 24-well plates, and then were treated with LPS (200 ng/ml), Pam3Csk4 (1 μg/ml) and CpG DNA(1 μg/ml). After a 72-h incubation at 37°C, cells were washed with PBS several times, and then resuspended in 100 μl of a binding buffer containing fluoroscein isothiocynate (FITC)-conjugated anti-AnnexinV antibody at room temperature for 15 min (BD Biosciences). Mixed with 400 μl binding buffer, the cells were analysed by flow cytometry (FACSCalibur; BD Biosciences) and the data were analysed with BD FCASDiva software (BD Bioscience).

Enzyme-linked immunosorbent assay (ELISA)

The IL6 in the supernatants obtained from purified MM cells and MM cell lines following TLR ligands treatment were measured using a standard enzyme-linked immunosorbent assay (ELISA) (R&D Systems). All experiments were performed at least three times.

Subcellular fractionation assay

Cytoplasm and nuclear fractions were obtained as described (Rahmouni et al, 2005). A total 6 × 106 cells were resuspended in ice-cold hypotonic buffer [42 mmol/l KCl, 10 mmol/l HEPES (pH 7·4), 5 mmol/l MgCl2, 1 mmol/l Na3VO4 and EDTA-free protease inhibitor cocktail], and incubated on ice for 15 min. Cells were then sheared by five passes through a 30-gauge needle. The lysates were centrifuged at 500 g for 10 min. The supernatant (cytosol) was collected and the pellet of nuclear material was washed three times in hypotonic buffer and then collected. Extracts were analysed with Western blotting using antibodies against NF-κB p65 subunit, PARP (nuclear) and tubulin (cytoplasmic).

Western blot analyses

Cell lysates were prepared in sodium dodecyl sulphate (SDS) sample buffer [62·5 mmol/l Tris/HCl (pH 6·8), 2% SDS, 10% glycerol, 50 mmol/l dithiothreitol, 0·1% bromophenol blue] containing a cocktail of protease inhibitors (Roche). Equal amounts of protein were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Roche). The membrane was blocked with PBST (0·05% Tween 20 in PBS) containing 5% skimmed milk and then incubated overnight with the primary antibody. Membranes were washed three times in PBST and then incubated with horseradish peroxidase-conjugated secondary antibody for 3–4 h. After further washing with PBST, proteins were visualized using an enhanced chemiluminescence Western blot system (Western Lightning; Perkin Elmer, Waltham, MA, USA).

Statistical analysis

Significance levels in TLRs expression were determined by non-parametric Mann–Whitney’s U-test for unpaired observations. Data were presented as mean ± standard error of the mean/standard deviation (SEM/SD) of several independent experiments. A P-value <0·05 was considered to be statistically significant, and was calculated with the Statistical Package for the Social Sciences (spss) software, version 13·0 (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Higher expression of TLR2, TLR4 and TLR9 mRNA in BMMCs of MM patients than those of healthy donors

To examine the transcript expression of TLRs in BMMCs from MM patients and their normal counterparts, the specific TLR1 – TLR10 transcripts in BMMCs from 29 MM patients at diagnosis and 25 healthy donors were measured with quantitative RT-PCR. The clinical characteristics of these patients are summarized in Table 1 and the sequences of primers used in this study are provided in Table S1. All TLR transcripts could be detected in BMMCs except for that of TLR3. The expression of TLR2, TLR4, and TLR9 in MM patients was significantly higher than those in healthy donors (over two-fold; P < 0·05). The expression of TLR1, TLR6 and TLR7 was higher in MM patients than in healthy donors, however this was not significantly different (Fig 1A). Further flow cytometric measurement of TLR2, TLR4 and TLR9 expression in the thawed BMMCs from 10 patients and donors demonstrated that expression of TLR4 and TLR9 in MM patients was significantly higher than those in healthy donors, although the expression of TLR2 was not significantly different between the two groups (Fig 1B).

image

Figure 1.  Expression of TLR1 – TLR10 in MM patients and healthy donors and the effect of several TLR ligands on the growth of MM cells. (A) Expression of TLR1 – TLR10 transcripts in BMMCs from 29 MM patients and 25 healthy donors was determined by quantitative RT-PCR and the relative expression of each TLR transcript normalized to that of GAPDH was presented as mean ± standard error of the mean (mean ± SEM). (B) Expression of TLR2, 4 and 9 on the surface of freshly thawed BMMCs from 10 MM patients and 10 healthy donors were analysed with flow cytometry and the MFI (Mean Fluorescence Intensity) of each TLR were compared between patients and donors. (C) 5 × 105 isolated BMMCs from three individual MM patients were treated with Pam3Csk4 (1 μg/ml), LPS (200 ng/ml) and CpG DNA (1 μg/ml) for 72 h. The cells were then analysed with anti-human CD138 antibody by flow cytometry. (D) Change of the proportion of CD138+ cells from 10 patients and five donors upon various treatments were normalized to those of the mock cells. P values were calculated with Mann–Whitney’s U-test. ★★< 0·01, ★< 0·05. MM, multiple myeloma; BMMCs, bone marrow mononuclear cells.

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LPS and CpG DNA could increase the CD138+ cells among the BMMCs of MM patients

To determine whether the BMMCs from MM patients and healthy donors respond differently to the ligands of TLR2, TLR4 and TLR9, the freshly separated BMMCs of these two groups were cultured with Pam3Csk4 (TLR2 ligand), LPS (TLR4 ligand), and CpG DNA (TLR9 ligand) respectively, for 72 h. The proportion of CD138-positive cells following TLR ligand treatment was compared with that of the untreated control cells. Three representative data showing CD138 expression of the ligand-stimulated cells, analysed by flow cytometry, are shown in Fig 1C; the response of cells from MM patients (n = 10) and healthy donors (n = 5) are summarized in Fig 1D. The data showed that LPS and CpG DNA stimulation increased the proportion of CD138-positive cells by nearly 60%, whereas Pam3Csk4 did not have similar effect on these cells. None of the TLR ligands altered the proportion of CD138-positive cells from the healthy donors. This observation suggested that the specific TLR ligands might increase the growth of MM cells.

TLR transcript expression in MM cells and normal plasma cells

To examine TLR transcript expression in MM cells and their normal counterparts, CD138+ cells from five MM patients and three healthy donors were purified with microbeads to yield a purity of over 90% (Fig 2B). The expression of TLR transcripts of these five primary MM samples, together with two MM cell lines U266 and RPMI-8226 and three normal plasma cells, were analysed by RT-PCR (Fig 2A). TLR transcripts could be detected in most of these five patients; in addition, TLR4, TLR7, TLR8 and TLR9 were detectable in all patients. In contrast, few TLR mRNA were detected in normal plasma cells. TLR4 and TLR9 were detectable in all five patients and the two cell lines; however TLR9 was only detected in one out three healthy donors.

image

Figure 2.  The repertoire of TLRs in primary myeloma cells from MM patients, MM cells lines and normal plasma cells. (A) Primary myeloma cells from five MM patients and normal bone marrow mononuclear cells (BMMC) from three healthy donors (HD) were purified with CD138 microbeads according to the manufacturer’s instructions, the obtained cells together with U266 and RPMI 8226 cells were used for the total RNA extraction. The expression of transcripts of TLR1 - TLR10 was analysed with conventional RT-PCR, human-peripheral blood mononuclear cells (PBMCs) and normal human-B cells served as the control. (B) A representative flow cytometry profile of CD138 enriched primary myeloma cells or normal PBMCs. All the purified cells achieved the purity over 90%.

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LPS and CpG DNA promoted MM cells proliferation and survival

To study the effect of LPS and CpG DNA directly on MM cells proliferation, we used CD138 purified cells from two MM patients at diagnosis and RPMI-8226 cell line as the target cells, meanwhile normal plasma cells were used as the control. All the cells were treated with Pam3Csk4, LPS and CpG DNA respectively, for 24, 48 and 72 h, and cell proliferation was measured by MTT assay. LPS and CpG DNA were shown to promote cell growth of MM cells compared to untreated cells; however Pam3Csk4 did not consistently promote the growth of these cells. Interestingly, the cell growth of the normal plasma cells could not be enhanced by treatment with any TLR ligands (Fig 3A).

imageimage

Figure 3.  The effect of LPS and CpG DNA on MM cell proliferation and survival. (A) Primary cells from two MM patients (MM$, MM%), RPMI-8226 cells (IL6-independent), XG-7 cells (IL6-dependent) and normal plasma cells were treated with Pam3Csk4 (1 μg/ml), LPS (200 ng/ml) and CpG DNA (1 μg/ml) for 24, 48 and 72 h, and the resulting cell proliferation measured by MTT assay. The relative cell expansion was plotted. (B) Primary cells from two MM patients, RPMI-8226 cells, XG-7 cells were treated with Pam3Csk4 (1 μg/ml), LPS (200 ng/ml) and CpG DNA (1 μg/ml) for 72 h, and the resulting cell apoptosis was assessed with Annexin V antibody using flow cytometry. (C) The production of IL6 in the supernatant of primary cells from two MM patients, RPMI-8226 cells, XG-7 cells (fresh media without additional rIL6 supplement was used for this measurement) before and after TLR ligand treatment was measured by ELISA. (D) Proliferation of primary cells from two MM patients, RPMI-8226 cells (IL6-independent) and XG-7 cells (rIL6 was previously removed from the media) under various conditions was measured by MTT assay 72 h later. The cells were treated with LPS (200 ng/ml), CpG DNA (1 μg/ml) and rIL6 (1 ng/ml) in the presence of normal IgG (10 μg/ml) or anti-IL6 (10 μg/ml) neutralizing antibodies as indicated in the panel; and the relative cell proliferation normalized to the un-treated cells was shown. ★mean < 0·05.

In order to study the effect of TLR ligands on MM cell survival, we first induced the apoptosis of these MM cells with serum-deprivation. As indicated previously (Le Gouill et al, 2004), these cells were starved in media supplemented with only 2% serum (compared to 15% for maintenance) in the absence or presence of Pam3Csk4, LPS and CpG DNA respectively, for 72 h, and then cell apoptosis was analysed with Annexin V antibody. The apoptosis caused by serum-deprivation was dramatically reduced with LPS and CpG treatments, whereas Pam3Csk4 showed less effect on protecting these cells from apoptosis (Fig 3B). In contrast, none of these TLR ligands rescued the apoptosis of normal plasma cells caused by serum-deprivation.

Induced IL6 production following TLR ligand treatment partially contributes to MM cell proliferation

It was reported that LPS and other TLR ligands could strongly induce the production of many cytokines including IL6. Previous reports had suggested that IL6 might be the target gene for TLRs to increase MM cell growth (Bohnhorst et al, 2006). We then measured IL6 production in supernatants of CD138-enriched MM cells from two patients, RMPI-8226 (IL6-independent) and XG-7 cells (IL6-dependent) before and after LPS and CpG DNA treatment, with ELISA. IL6 production was dramatically increased upon TLR ligand treatment when compared to untreated cells (Fig 3C). In addition, purified MM cells and XG-7 cells produced more IL6 than RPMI-8226 cells after the ligand stimulation, which might suggest that IL6 was a key cytokine involved in the promotion of MM cell growth with the treatment of TLR ligands. Interestingly, the IL6 neutralizing antibody partially inhibited LPS and CpG DNA-induced cell proliferation of MM cells, RPMI-8226 and XG-7 cells (Fig 3D), while the isotype control IgG did not abolish ligand-induced proliferation. These data indicated that the increased proliferation in MM cells and MM cell lines following TLR ligand treatment was partially due to IL6autocrine.

LPS and CpG DNA reversed the suppression of MM cell growth caused by blockage of MAPK and JNK signalling pathways but not NF-κB

Triggered by the binding of TLR ligands, TLRs would activate the downstream signalling pathways, including MAPK, JNK and NF-κB. To explore whether, and to what extent, these signalling pathways were involved in the increased MM cell proliferation induced by LPS and CpG DNA, we firstly inhibited each of them with either small molecules or inhibitory plasmid. JNK-I (SP600125), PD169316 and PD9805 inhibited JNK, p38 and ERK, while plasmid IκBα-SR encoded a superrepressor form of IκBα. The growth of MM cells were reduced by all these inhibitory agents; LPS and CpG DNA were able to reverse the JNK, p38 and ERK inhibitor suppressed cell growth but not the IκBα suppressed cell growth, which indicated a crucial role of activated NF-κB in increased MM cell growth by TLR ligands (Fig 4).

image

Figure 4.  The effect of inhibition of MAPK, JNK and NF-κB signalling pathways on MM cells growth following LPS and CpG DNA treatments. (A) To target MAPK, JNK and NF-κB signalling pathways, U266 and RPMI 8226 cells were incubated with PD169316 (10 μmol/l), JNK-I (10 mmol/l) and PD98059 (10 μmol/l) or transiently transfected with 0·2 μg IκBα-SR respectively for 72 h, and then cell proliferation with or without LPS (200 ng/ml) was measured by MTT assay. The relative growth compared to the untreated cells is presented. (B) To target MAPK, JNK and NF-κB signalling pathways, U266 and RPMI 8226 cells were incubated with PD169316 (10 μmol/l), JNK-I (10 mmol/l) and PD98059 (10 μmol/l) or transiently transfected with 0·2 μg IκBα-SR for 72 h, and then cell proliferation with or without CpG DNA(1 μg/ml) was measured by MTT assay. The relative growth compared to the untreated cells was presented. ★mean < 0·05.

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Activation of NF-κB by LPS and CpG DNA contributed to MM cells growth

Nuclear translocation of the NF-κB p65 subunit was a crucial step of the activation of canonical NF-κB pathway. Thus we analysed the subcellular localization of NF-κB p65 subunit upon ligand stimulation and found that nuclear translocation of the p65 subunit took place when cells were treated with ligands (Fig 5A). Next, we explored whether the activation of NF-κB alone was sufficient to promote the growth and IL6 transcription of U266 and RPMI-8226 cells. IKKα and IKKβ were delivered into U266 and RPMI-8226 cells and, as expected, the p65 subunit transferred into nucleus (Fig 5A). The forced expression of IKKα and IKKβ promoted cell growth and increased IL6 transcript in a dose-dependent manner (Fig 5B, C). In contrast, the inhibitory plasmid IκBα-SR alone suppressed both cell proliferation and IL6 transcript expression of these cells (Fig 5B, C). Taken together, these data might suggest that LPS and CpG DNA triggered the activation of TLRs through binding, and then the activated canonical NF-κB resulted in increased IL6 transcription, whose protein product was partially responsible for the MM cell growth and survival (Fig 5D).

image

Figure 5.  Activation of NF-κB by LPS and CpG DNA contributed to MM cells growth. (A) U266 cells were treated with LPS (200 ng/ml), CpG DNA (1 μg/ml) or transfected with 0·4 μg IKKα/IKKβ plasmids for 24 h respectively. Cytoplasmic and nuclear fractions were isolated and subjected to Western blot analysis using antibodies against NF-κB p65, tubulin (cytoplasm) and PARP (nuclear). (B) U266 cells and RMPI 8226 cells were transiently transfected with 0·2 μg IκBα-SR and various doses of IKKα/IKKβ. Cell proliferation was measured by MTT assay 72 h after transfection and the relative growth compared with un-transfected cells was plotted. (C) With same treatment described in panel B, IL6 mRNA levels were measured with quantitative RT-PCR, the relative expression of IL6 normalized to that of GAPDH of cells with various treatments was presented. (D) Schematic illustration of LPS and CpG DNA triggered TLR4 and TLR9 activation, which stimulated the IL6 production via canonical NF-κB signalling to enhance the MM cell proliferation and protect them from apoptosis. ★mean < 0·05.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Toll-like receptors (TLRs) act as the sensors for the innate immunity and initiators of adaptive immunity, and are involved in the activation of normal B lymphocytes. Human B cells are characterized by high expression of TLRs 1, 5, 6, 7, 9 and 10 (Hornung et al, 2002; Bernasconi et al, 2003; Bourke et al, 2003), and the inability to be activated by LPS due to the lack of TLR4. Recent reports have described the importance of the TLR system in normal and malignant B cells proliferation, differentiation and anti-apoptotic effects (Chiron et al, 2008). MM is a clonal plasma cell malignancy, however little is currently known about the repertoire and function of TLRs in MM cells.

One study found that only a few TLRs (mainly TLR1) were expressed in plasma cells from BM of healthy donors while frequent TLR expression was detected in cell lines generated from MM patients, and TLR-specific ligands could induce the increased proliferation and survival of these MM cell lines (Bohnhorst et al, 2006). Meanwhile, Jego et al (2006) also reported that human myeloma cell lines (HMCL) and primary myeloma cells expressed a broad range of TLRs, and culture with TLR7 and TLR9 ligands saved HMCL from serum-deprivation or dexamethasone-induced apoptosis. The present study showed that all TLRs, except TLR3, were present in most BMMCs from MM patients. Interestingly we also found that transcript expression of TLR2, TLR4 and TLR9 were higher in BMMCs of newly diagnosed MM patients compared with those of healthy donors. Most TLR mRNA could be detected in CD138 purified cells from MM patients, and expression of TLR2, TLR4, TLR7 and TLR9 was present in all MM plasma cells used in this study. In addition, TLR4 and TLR9 were more highly expressed in BMMCs from MM patients than healthy donors. These data are similar to previous reports(Bohnhorst et al, 2006; Jego et al, 2006); we also detected a small difference in TLR expression profiles in MM cells compared to previous reports, which might be due to the variation among patients.

It is well established that IL6 acts as a primary growth factor for freshly isolated myeloma plasma cells as well as myeloma cell lines (Klein et al, 1990), and previous reports had suggested that IL6 might be a target gene for TLRs to promote the growth of MM cells, despite the fact that some MM cell lines proliferated in an IL6 independent fashion (Bohnhorst et al, 2006). In the present study, TLR4 and TLR9 ligands promoted MM cell growth and survival, which was partially due to increased autocrine production of IL6. Though MAPK and canonical NF-κB were both documented possibly to be activated through TLRs (Akira, 2003; Kawai & Akira, 2006), the present data suggested a specific requirement of activation of NF-κB for TLR ligand-promoted proliferation and survival. Interestingly, the activation and inhibition of canonical NF-κB in two MM cell lines consistently enhanced or reduced the transcript expression of IL6. It is plausible to hypothesize that upregulation of IL6 through NF-κB following TLR ligand stimulation might be responsible for such promotion of MM cell proliferation and survival (Fig 5D).

Previous reports found that TLR1, TLR2, TLR5, TLR7 and TLR9 had the potential to enhance MM cell proliferation and protect MM cells from apoptosis (Bohnhorst et al, 2006; Jego et al, 2006), which suggested this property could be a common feature of the TLR family and not restricted to only TLR4 and TLR9, focused in present study. However as TLR 4 and TLR9 were differentially expressed between malignant and normal cells, an in depth mechanistic study might provide novel therapeutic targets against MM.

TLR 4 and TLR 9 shared the same signalling pathways, however there were several differences between TLR4 and TLR9. For example, stimulation of TLR4 facilitated the myeloid differentiation primary-response protein 88(MyD88)-dependent and MyD88-independent pathway, while TLR9 mainly induced inflammatory cytokines and type I interferon via MyD88-dependent pathway (Akira & Takeda, 2004). In this study, activated TLR4 and TLR9 were shown to promote MM cell growth, which suggested the activation of NF-κB was triggered by TLR ligands mainly through the MyD88 dependent pathway. Further experiments to clarify the role of MyD88 in TLR4- and TLR9x- mediated signal pathways is of great interest.

The BM microenvironment is known to contribute substantially to the malignant growth and survival of MM cells (Caligaris-Cappio et al, 1992; Hallek et al, 1998). In fact, bone marrow stromal cells (BMSCs), in particular, provide a number of cytokines that might contribute to the growth of myeloma cells. Adhesion of MM cells to BMSCs not only localizes MM cells in the marrow microenvironment, but also triggers IL6 secretion by BMSCs and regulates MM cell proliferation (Uchiyama et al, 1993). It was reported that NF-κB activation was important in regulation of IL6 transcription that was triggered in BMSCs after MM cell adhesion (Chauhan et al, 1996). To date, little is known about the relationship between BMSCs and TLRs. Nevertheless, it would be of interest to explore the relationship between TLRs, BMSCs and MM cells in next step to challenge the hypothesis that TLR ligands could not only favour MM cell proliferation directly but also help BMSCs to induce production of cytokines, including IL6, and subsequently promote MM cells growth.

Most of the research in B-chronic lymphocyte leukaemia (B-CLL) has concentrated on TLR7 and TLR9, as preliminary data suggested that they are the two main TLRs expressed on CLL cells (Decker et al, 2002; Spaner et al, 2006; Grandjenette et al, 2007; Muzio et al, 2009). TLR7 and TLR9 agonists may indirectly clear CLL cells by enhancing the activity of natural killer and tumour-reactive T cells, or by altering the tumour microenvironment and inhibiting angiogenesis (Spaner & Masellis, 2007). Current research has enabled the use of these agents to strengthen the effects of standard chemotherapy and irradiation therapy (McGettrick & O’Neill, 2007). However, the role of TLRs in MM remains unclear. In healthy donors, microbe pathogens promote plasma cell differentiation and antibody production, whereas in MM patients, stimulation of microbe pathogen would favour the development of normal plasma cells and malignant myeloma cells. Recent epidemiological studies showed that several episodes of pneumonia prior to MM diagnosis increased the risk of developing MM (Landgren et al, 2006). In combination with other co-factors, constant stimulation may initiate or promote tumour formation. In addition, the interaction between TLRs and ligands would favour escape from standard therapies (Chiron et al, 2008). Therefore, it is possible that the TLR system could be developed as a therapy target in the future, as interference of the TLR signalling pathway could limit tumour formation and provide an adjuvant therapy to standard treatments.

Taken together, this study demonstrated that expression of TLRs 2, 4 and 9 were higher in BMMCs of newly diagnosed MM patients than their normal counterparts; in addition TLR4 and TLR9 were also more highly expressed in purified MM cells from MM patients. Consequently, TLR4- and TLR9-specific ligands could promote MM cell proliferation and prevent MM cells from serum-deprivation induced apoptosis. Induced IL6 production was responsible for MM cell growth following TLR ligand treatment, which was possibly actionedvia the activation of the canonical NF-κB cascade.

Funding

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

This work was supported by research grant LJ200626 from Scientific Research Foundation of Health Ministry of China, and WKF07003 from Clinical Medicine Centre Haematology Project of Jiangsu Province.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Conflict of interest
  7. Funding
  8. References
  9. Supporting Information

Table SI. The primer sequences.

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BJH_8284_sm_Table-SI.doc45KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.