research paper
Expression and function of toll-like receptors in multiple myeloma patients: toll-like receptor ligands promote multiple myeloma cell growth and survival via activation of nuclear factor-jB
Yang Xu,1 Yun Zhao,2 Haiwen Huang,1 Guanghua Chen,1 Xiaojin Wu,1 Ying Wang,1 Weirong Chang,1 Ziling Zhu,1 Yufeng Feng1 and Depei Wu1,2 1
The First Affiliated Hospital of Soochow University, Jiangsu Institute of Haematology, Key laboratory of Thrombosis and Haemostasis, Ministry of Health, Suzhou, Jiangsu Province, and
2
Cyrus Tang Haematology Centre, Jiangsu
Institute of Haematology, the First Affiliated Hospital, Soochow University, Suzhou, Jiangsu Province, China
Received 21 December 2009; accepted for publication 4 May 2010 Correspondence: Depei Wu, The First Affiliated Hospital of Soochow University, Jiangsu Institute of Haematology, Key laboratory of Thrombosis and Haemostasis, Ministry of Health, Suzhou, Jiangsu Province, China,
Summary 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-jB activities. Further investigation demonstrated that these TLR ligands could trigger the nuclear translocation of NF-jB p65 and the activated NF-jB was sufficient to increase the expression of IL6 transcript in MM cells. These data suggested that activated NF-jB 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.
215006. E-mail:
[email protected] Yang Xu and Yun Zhao contributed equally to this manuscript.
Keywords: Multiple myeloma, Toll-like receptor, Toll-like receptor ligands, interleukin-6, nuclear factor-kappa B.
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 a (TNF-a) (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-jB) and interferon regulatory factors (IRFs), which eventually result in the
ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 150, 543–553
First published online 14 July 2010 doi:10.1111/j.1365-2141.2010.08284.x
Y. Xu et al 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.
1 ng/ml recombinant IL6 (rIL6). Cells were cultured at 37C in a humidified 5% CO2 atmosphere.
Chemicals, antibodies, plasmids and transfections
Parameter
Patients
Healthy donors
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-jB p65 was from Cell Signaling Technology (Danvers, MA, USA); and peroxidaseconjugated 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 RcCMVIjBa-SR (IjBa-SR) expressed a superrepressor form of IjBa, whose serines 32 and 36 were mutated to alanines (Traenckner et al, 1995). Plasmids pRc-b-actin3HA-IKKa and –IKKb (IKKa/b) 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 b-galactosidase in the cell lysates (b-Galactosidase Enzyme Assay kitC; Promega, Madison, WI, USA) after co-transfection with the pSV-b-galactosidase (Promega) vector as the control.
Number (N/R) Median age (range), years Male/female Type
29 (27/2) 59 (33–78)
25 48 (22–64)
RNA extraction and real-time PCR
16/13 IgG 8; IgA 2; IgG,j 4; IgA,j 2; IgD,k 1; IgD,j 1; k 2; (–) 9 II A 5/20; II B 1/20; III A 6/20; III B 8/20 Lung 4/20; CRF 9/20; Anaemia 9/20; Osteolysis 9/20 36Æ1(7Æ5–62Æ5)
13/12 Normal 19; IDA 6
Materials and methods 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 Table I. Clinical features of MM patients and healthy donors.
Stage (Durie–Salmon) Organs involved
Average plasma cells in BM at diagnosis (%)
1Æ1(0Æ5–2Æ0)
(–): No data. N/R, newly diagnosed/relapsed; BM, bone marrow; IDA, iron deficiency anaemia; CRF, chronic renal failure; Lung, lung infection.
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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 SuperscriptTM 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 TaqTM (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 ll. After an initial denaturation step at 95C for 5 min, 45 cycles of a three-step PCR with a single fluorescence measurement were undertaken (95C for 15 s, 60C for 30 s, and 72C for
ª 2010 Blackwell Publishing Ltd, British Journal of Haematology, 150, 543–553
Expression and Function of TLRs in MM Patients 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).
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%.
Flow cytometry Freshly thawed BMMCs from patients and healthy donors were enumerated, and then 500 ll 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).
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 lg/ml) and CpG DNA(1 lg/ml). After a 72-h incubation at 37C, cells were washed with PBS several times, and then resuspended in 100 ll of a binding buffer containing fluoroscein isothiocynate (FITC)-conjugated anti-AnnexinV antibody at room temperature for 15 min (BD Biosciences). Mixed with 400 ll 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) 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 lg/ml) and CpG DNA (1 lg/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 ll RPMI 1640 media were plated and then treated with vehicle, LPS (200 ng/ml), Pam3Csk4 (1 lg/ml) and CpG DNA (1 lg/ml) respectively, for 24, 48 and 72 h. After treatment, 20 ll of 5 mg/ml MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 4 h at 37C. The plate was then spun at 1000 g for 5 min and the media were removed. The remaining cells were solubilized with 100 ll 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
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 7Æ4), 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-jB p65 subunit, PARP (nuclear) and tubulin (cytoplasmic).
Western blot analyses Cell lysates were prepared in sodium dodecyl sulphate (SDS) sample buffer [62Æ5 mmol/l Tris/HCl (pH 6Æ8), 2% SDS, 10% glycerol, 50 mmol/l dithiothreitol, 0Æ1% 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 (0Æ05% Tween 20 in PBS) containing 5% skimmed milk and then incubated overnight
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Y. Xu et al 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
