Polymorphisms in the interleukin-1 receptor antagonist and interleukin-6 genes affect risk of osteolysis in patients with total hip arthroplasty.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 58, No. 10, October 2008, pp 3157–3165 DOI 10.1002/art.23863 © 2008, American College of Rheumatology Polymorphisms in the Interleukin-1 Receptor Antagonist and Interleukin-6 Genes Affect Risk of Osteolysis in Patients With Total Hip Arthroplasty Andrew Gordon,1 E. Kiss-Toth,1 Ian Stockley,2 Richard Eastell,1 and J. Mark Wilkinson1 ciated with osteolysis. The uncommon IL6 haplotype ⴚ174G/ⴚ572G/ⴚ597A (osteolysis group frequency 2.4%, control group frequency 0.8%) was associated with osteolysis (P ⴝ 0.02, calculated using Haploview software). The IL1RA ⴙ2018CC genotype was associated with increased mRNA expression compared with the ⴙ2018TT genotype in both unstimulated and stimulated PBMCs (P ⴝ 0.01 by analysis of variance, after Bonferroni correction). Conclusion. The IL1RA ⴙ2018C allele is associated with a decreased risk of osteolysis after THA and with increased IL-1 receptor antagonist mRNA expression in vitro. An uncommon haplotype within the promoter region of the gene for IL-6 is positively associated with osteolysis. Objective. To determine whether osteolysis after total hip arthroplasty (THA) is associated with common polymorphisms within the genes encoding the interleukin-1 (IL-1) family and IL-6, and to determine whether polymorphisms that are associated with osteolysis affect in vitro messenger RNA (mRNA) expression in human peripheral blood mononuclear cells (PBMCs) in response to wear particles. Methods. Unrelated white subjects of North European descent (n ⴝ 612) were recruited a mean of 11 years after cemented THA for primary osteoarthritis. Of these subjects, 272 had previous osteolysis and 340 had no radiographic evidence of osteolysis (control group). Genomic DNA was genotyped for the following singlenucleotide polymorphisms (SNPs): IL1A ⴙ4845, IL1B ⴙ3954, IL1B –3737, IL1B –511, IL1RA ⴙ2018, IL6 –174, IL6 –572, and IL6 –597. In a subset of 60 subjects, PBMCs were extracted and stimulated with titanium particles and/or endotoxin, and cytokine mRNA expression was measured using quantitative real-time reverse transcriptase–polymerase chain reaction. Results. The odds ratio (OR) for osteolysis associated with carriage of the IL1RA ⴙ2018C allele was 0.66 (95% confidence interval [95% CI] 0.48–0.91) (P ⴝ 0.012). The remaining SNPs were not individually asso- Particulate wear debris is implicated as a major factor contributing to osteolysis and aseptic loosening after total hip arthroplasty (THA) (1). In this model, macrophages in the periprosthetic environment express proinflammatory cytokines and enzymes in response to implant-derived wear particles. Both in vitro and in vivo evidence shows that wear debris–stimulated macrophages release interleukin-1 (IL-1) family and IL-6 cytokines, and these cytokines are found at high levels in osteolytic tissues (2–6), synovial fluid (7), and regional lymph nodes retrieved from failed THAs, while they are not found in stable implants (8). These cytokines are known to mediate bone resorption, both in vivo and in vitro. The proinflammatory cytokines IL-1␣ and IL-1␤ bind to IL-1 receptor type I (IL-1RI) and initiate cellular signaling. Both IL-1␣ and IL-1␤ induce increased osteoclast formation and activation, effects that are mediated through RANKL (9,10). IL-1R antagonist (IL-1Ra) also binds to IL-1RI and acts as a competitive antagonist to signal transduction (11). IL-6 binds to the IL-6 receptor, consisting of a signal transducer and a co- Supported by the Royal College of Surgeons of England, the Arthritis Research Campaign, the Cavendish Hip Foundation, and the John Charnley Trust. 1 Andrew Gordon, MB ChB, MRCS, E. Kiss-Toth, PhD, Richard Eastell, MD, FRCP, J. Mark Wilkinson, PhD, FRCS: University of Sheffield, Sheffield, UK; 2Ian Stockley, MD, FRCS: Northern General Hospital, Sheffield, UK. Dr. Eastell owns stock or stock options in Interleukin Genetics. Address correspondence and reprint requests to J. Mark Wilkinson, PhD, FRCS, Academic Unit of Bone Metabolism, Sorby Wing, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK. E-mail: firstname.lastname@example.org. Submitted for publication March 16, 2008; accepted in revised form June 6, 2008. 3157 3158 GORDON ET AL receptor chain, and initiates downstream signaling that results in osteoclast formation, although its osteoclastogenic effect is weaker than that of IL-1 (12,13). The development of osteolysis after THA is influenced in part by environmental factors (14). However, much of the observed variation between patients in susceptibility to osteolysis remains unexplained and may be influenced by genetic factors. Several polymorphisms within the genes encoding the IL-1 family and IL-6 have been described (http://www.ncbi.nlm.nih.gov/snp/). These polymorphisms are associated with differences in susceptibility to infectious and inflammatory conditions and disorders of bone, including decreased bone mineral density, rheumatoid arthritis (RA), osteoarthritis (OA), and periodontitis (15–20). Recently, polymorphisms within the genes encoding tumor necrosis factor, IL-6, and transforming growth factor ␤, as well as within wnt signaling genes, have been associated with osteolysis after THA (21–23). However, the potential association with osteolysis of polymorphisms within the genes encoding the IL-1 family remains unexplored, and polymorphisms within the gene encoding IL-6 have, to date, been studied only in a small subject population (22). At a cellular level, DNA variation may affect gene transcription, messenger RNA (mRNA) stability, protein quantity, and protein function. However, the function of these polymorphisms in the setting of osteolysis also remains unclear. One aim of this study was to determine whether there is an association between osteolysis after THA and common polymorphisms in the genes encoding the IL-1 family and IL-6. Using quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR), we further aimed to determine whether polymorphisms significantly associated with osteolysis in our population affect in vitro mRNA expression in human peripheral blood mononuclear cells (PBMCs) in response to wear particles. SUBJECTS AND METHODS Case–control association study. Study subjects and phenotyping. The study was approved by the local research ethics committee, and all subjects provided written, informed consent prior to participation. White subjects of North European descent were recruited. All had a diagnosis of idiopathic OA of the hip and had previously received a cemented THA with a polyethylene-on-metal bearing couple. The exclusion criteria have been reported elsewhere (21). The subjects were divided into 2 phenotype groups: an osteolysis group and a control group. The osteolysis group comprised subjects with current radiographic evidence of osteolysis and subjects who had previously undergone revision for confirmed aseptic loosening. The criteria of Harris and McGann and those of Harris and Penenberg were used to define loosening of the femoral or pelvic component, respectively (24,25). Control group subjects had no current evidence of osteolysis on plain anteroposterior and lateral radiographs of the hip obtained on the day of genotyping. The EBRA-Digital method (University of Innsbruck, Innsbruck, Austria) was used to measure acetabular polyethylene wear on digitized anteroposterior radiographs of the pelvis, according to a protocol described previously (26). In the osteolysis subjects, measurements were made using the radiograph obtained immediately prior to revision surgery, and in the control subjects, the radiograph obtained on the day of genotyping was used. Single-nucleotide polymorphism (SNP) selection and genotyping. Genomic DNA was extracted from whole blood using the chloroform–ethanol method. Candidate SNPs within the genes encoding IL-1␣, IL-1␤, IL-1Ra, and IL-6 were selected using information available in the public databases dbSNP (http://www.ncbi.nlm.nih.gov/snp/) and HapMap (http://www.hapmap.org/) according to the following criteria: 1) validated SNPs, 2) minor allele frequency in European populations ⬎5%, and 3) reported data showing association with bone diseases from at least 2 independent sources. DNA was genotyped for the SNPs rs17561 (IL1A ⫹4845), rs4848306 (IL1B ⫺3737), rs16944 (IL1B ⫺511), rs1143634 (IL1B ⫹3954), rs419598 (IL1RA ⫹2018), and rs1800795 (IL6 ⫺174), using the TaqMan 5⬘ nuclease method (Applied Biosystems, Warrington, UK). Probes and primer sequences are listed in Table 1. The SNPs rs1800796 (IL6 ⫺572) and rs1800797 (IL6 ⫺597) were assayed using TaqMan Assay on Demand minor groove binder probes and primers (Applied Biosystems) (Table 1). The TaqMan 5⬘ nuclease method reaction mixture contained 1⫻ Taq polymerase and 0.8 ng/l of DNA template in a final volume of 15 l. The primer concentration varied between 100 nM and 500 nM, and the probe concentration varied between 50 nM and 100 nM, following assay optimization. The PCR conditions were heating to 50°C for 2 minutes, then 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 61°C for 1 minute. The TaqMan Assay on Demand reaction mixture and PCR conditions were prepared following the manufacturer’s instructions. PCR was performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA), and fluorescence detection and genotype identification were performed using an ABI Prism 7900HT sequence detector (Applied Biosystems). Repeat genotyping for quality control in 10% of the subjects revealed 100% concordance. PBMC stimulation study. Subject selection and phenotyping. This study received separate approval from the local research ethics committee. All subjects provided written, informed consent prior to participation. We selected a subgroup of subjects who had participated in both the control and osteolysis groups of the genotyping population. The exclusion criteria for this study were any history of active or recent viral/bacterial infections within the preceding month, current use of antiinflammatory or immunomodulatory agents, any history of chronic inflammatory disorders, and current radiographic evidence of osteolysis. Subjects who had previously had a revision procedure for osteolysis were recruited at least 1 RISK OF OSTEOLYSIS CONFERRED BY POLYMORPHISMS IN THA 3159 Table 1. Sequences of probes and primers for the IL1 gene family SNPs and for RT-PCR* SNP ID no., site rs17561, IL1A ⫹4845 rs4848306, IL1B ⫺3737 rs16944, IL1B ⫺511 rs1143634, IL1B ⫹3954 rs419598, IL1RA ⫹2018 rs1800795, IL6 ⫺174 Cytokine IL-1Ra ␤-actin Primer sequence Probe sequence TGCACTTGTGATCATGGTTTTAGA TCCTCATAAAGTTGTATTTCACATTGC CTAAGCCCTCCTTGTTCTAGACCA CTCTTGGATCCAGGTTCTCAGATT TTGAGGGTGTGGGTCTCTACCT AGGAGCCTGAACCCTGCATAC GCCTGCCCTTCTGATTTTATACC CATCGTGCACATAAGCCTCGTTA GGGATGTTAACCAGAAGACCTTCTATCT CAACCACTCACCTTCTAAATTGACATT GCTGATTGGAAACCTTATTAAGATTGT AATGACGACCTAAGCTGCACTTT AAGCCTAGGTCAGCACCTTTTAGCTTCC CAAGCCTAGGTCATCACCTTTTAGCTTCC AATGGAATGTCCCTTGGACTCTGCAT AATGGAATGTTCCTTGGACTCTGCATG TTCTCTGCCTCAGGAGCTCTCTGTCA TTCTCTGCCTCGGGAGCTCTCTGT CAGAACCTATCTTCTTCGACACATGGGA TTCAGAACCTATCTTCTTTGACACATGGGA ACAACCAACTAGTTGCCGGATACTTGC AACAACCAACTAGTTGCTGGATACTTGCAA ACGTCCTTTAGCATGGCAAGACACAAC ACGTCCTTTAGCATCGCAAGACACAAC TTGAGGGTGTGGGTCTCTACCT AGGAGCCTGAACCCTGCATAC GGGATGTTAACCAGAAGACCTTCTATCT CAACCACTCACCTTCTAAATTGACATT TTCTCTGCCTCGGGAGCTCTCTGT AACAACCAACTAGTTGCTGGATACTTGCAA * Shown are oligonucleotide sequences for TaqMan genotyping (for single-nucleotide polymorphisms [SNPs]) and for TaqMan reverse transcriptase–polymerase chain reaction (RT-PCR) (for cytokines). Forward primers are shown above reverse primers in primer sequences; TET probes are shown above FAM probes in probe sequences. The SNPs rs1800796 (IL6 ⫺572) and rs1800797 (IL6 ⫺597) were assayed using TaqMan Assay on Demand minor groove binder probes and primers (Applied Biosystems, Warrington, UK). The context sequence for SNP rs1800796 (Applied Biosystems assay ID no. C-11326893-10) was ATGGCCAGGCAGTTCTACAACAGCC[C/G]CTCACAGGGAGAGCCAGAACACAGA. The context sequence for SNP rs1800797 (Applied Biosystems assay ID no. C-1839695-20) was TGAAGTAACTGCACGAAATTTGAGG[A/G]TGGCCAGGCAGTTCTACAACAGCCG. IL-1Ra ⫽ interleukin-1 receptor antagonist. year following any surgical intervention and were free from any surgical complications. Investigators were blinded to individual genotypes at the time of subject selection. PBMC collection and preparation. Peripheral blood was obtained from all subjects between the hours of 9 AM and 11 AM following an overnight fast in order to standardize collection conditions. Thirty milliliters of peripheral blood was diluted 1:2 with Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Paisley, UK) and layered onto sucrose solution (Lymphoprep; Axis-Shield, Rodeløkk, Norway). PBMCs were collected after centrifugation at 800g for 20 minutes at 4°C. PBMCs were washed twice by suspension in DMEM and centrifugation at 350g for 10 minutes and 5 minutes, respectively. Following the final wash, cells were suspended in complete growth mixture consisting of DMEM containing 10% fetal calf serum, 5% L-glutamine, and 1% penicillin/streptomycin. Particle preparation and PBMC stimulation. Particles were supplied as a gift from Dr. E. M. Greenfield (Case Western Reserve University, Cleveland, OH). Two types of titanium particles (Johnson Matthey, Ward Hill, MA) had been prepared according to protocols previously described (27). These included endotoxin-stripped particles (Ti) and commercially pure endotoxin-stripped particles with adherent lipopolysaccharide (LPS) added back (Ti plus LPS). The protocols for preparation of the 2 types of particles are described in detail elsewhere, as are their size characteristics and their ability to induce cytokine production in mononuclear cells (28,29). High Sensitivity Limulus Amebocyte Lysate assay (BioWhittaker, Walkersville, MD) confirmed endotoxin levels of 0 EU/ml and 140 EU/ml for the Ti and Ti plus LPS particles, respectively. PBMCs were plated in duplicate in 96-well plates at a concentration of 5 ⫻ 106/well and were incubated for 2 hours (in 5% CO2 at 37°C) to allow sedimentation. Cell viability was confirmed by cell counting with trypan blue exclusion (97% viability was observed). PBMCs were then incubated with either Ti or Ti plus LPS particles at a concentration of 830 particles per cell. This particle-to-cell ratio had been previously found to give maximal mRNA expression with particle stimulation (data not shown). Cells stimulated with endotoxin (100 ng/ml LPS) and unstimulated cells acted as positive and negative controls, respectively. All stimulations were performed in duplicate. Cells were incubated for 3 hours in 5% CO2 at 37°C. A preliminary time-course experiment demonstrated that this time point gave the greatest discrimination for mRNA expression between LPS-stimulated and unstimulated cells (data not shown). RNA extraction and quantitation. Following stimulation, culture plates were spun at 2,000 revolutions per minute for 2 minutes at 4°C, and the cell supernatant was removed. The cells were then lysed and homogenized using QIA shredder columns (Qiagen, Crawley, UK), and total RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Total RNA was reverse transcribed using a high-capacity complementary DNA (cDNA) archive kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Primers and probes were synthesized by BioSource International (Camarillo, CA) and were designed using PrimerSelect LaserGene software (DNA Star, Madison, WI) (Table 1). Each quantitative real-time RT-PCR assay was performed in duplicate in a total volume of 15 l containing 1.5 l cDNA, one-step TaqMan quantitative real-time RT-PCR Master Mix, primers, and probes. PCR was 3160 GORDON ET AL Table 2. Characteristics of the genotype study subjects* Characteristic Control group (n ⫽ 340) Osteolysis group (n ⫽ 272) Age at primary THA, years No. of men/no. of women Body mass index, kg/m2 Annual wear rate, median (IQR) mm/year Osteolysis-free survival, years¶ 65 ⫾ 8 142/198 28 ⫾ 5 0.07 (0.05–0.10) 12 ⫾ 4 59 ⫾ 9 148/124 28 ⫾ 5 0.13 (0.08–0.21) 10 ⫾ 5 P ⬍0.001† ⬍0.001‡ ⬎0.05† ⬍0.001§ ⬍0.001§ * Except where indicated otherwise, values are the mean ⫾ SD. IQR ⫽ interquartile range. † By Student’s t-test. ‡ By chi-square test. § By Mann-Whitney U test. ¶ Osteolysis-free survival for the control group was the time from primary total hip arthroplasty (THA) surgery to the date of clinical review and genotyping; for the osteolysis group, it was the time from primary THA surgery to the date of diagnosis of osteolysis. performed on a PTC-200 thermal cycler. The PCR conditions were 95°C for 10 minutes, followed by 40 cycles of denaturing at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute. Detection of PCR products was performed using an ABI Prism 7900HT sequence detection system. Results were analyzed using ABI Prism SDS version 2.1 software and expressed as a ratio to ␤-actin mRNA expression. Study replication. Blood was obtained from all patients in duplicate, with a 1-week interval between each venesection. The experimental stimulation protocol was also followed in duplicate, with a 1-week interval between stimulations. Results for mRNA expression for each study were analyzed separately to confirm the robustness of the findings. Statistical analysis. All statistical analyses were 2-tailed and were made using SPSS statistical software (SPSS, Chicago, IL). Between-group comparisons were made using Student’s t-test for continuous normally distributed variables and the Mann-Whitney U test for continuous non-normally distributed variables. The chi-square test with Yates’ correction was used for analysis of allele carriage rates. Odds ratios (ORs) were calculated from counts of haplotypes estimated using the expectation-maximization algorithm in Haploview 4.0 (Broad Institute, Cambridge, MA). Results of the mRNA stimulation assays were analyzed using repeated-measures analysis of variance (ANOVA), with inclusion of revision status as a covariate. Messenger RNA expression levels for the replication study were compared using the Pearson correlation coefficient for each stimulation condition. RESULTS Case–control association study findings. Six hundred twelve subjects were recruited between February 2000 and April 2006, of whom 272 formed the osteolysis group. Within this group, 147 subjects (54%) had osteolysis around both the pelvic and femoral components, 69 subjects (25%) had osteolysis affecting only the pelvic component, and 56 subjects (21%) had femoral osteol- ysis only. Subjects in the osteolysis group were younger, a greater proportion were men, and they had a higher median annual rate of polyethylene wear compared with the control subjects (P ⬍ 0.05 for all comparisons) (Table 2). Implant wear was measured in 544 subjects (89%). For the remaining subjects, radiographs were unavailable or were of insufficient quality for wear measurements. Single SNP analysis. The distribution of genotypes for all SNPs within the control group showed them to be in Hardy-Weinberg equilibrium (P ⬎ 0.05 by chi-square test) (Table 3). In the osteolysis group, all SNPs were in Hardy-Weinberg equilibrium (P ⬎ 0.05 by chi-square test) except for SNP rs419598 (P ⫽ 0.01 by chi-square test) (Table 3). Repeat genotyping using TaqMan in 75% of this population and direct sequencing in a nonselective subgroup of 35 subjects showed 100% concordance with the original genotyping calls. The genotype distribution for SNP rs419598 differed between control and osteolysis groups (P ⫽ 0.007 by chi-square test). Carriage of the IL1RA ⫹2018C allele was underrepresented in the osteolysis group compared with the control group (P ⫽ 0.01 by chi-square test) (Table 3). The OR for osteolysis associated with carriage of the IL1RA ⫹2018C allele was 0.66 (95% confidence interval [95% CI] 0.48–0.91) (P ⫽ 0.012). After adjustment for the effects of other significant covariates (age, sex, polyethylene wear, and osteolysisfree survival) using multiple logistic regression analysis, the OR for osteolysis associated with carriage of the IL1RA ⫹2018C allele was 0.69 (95% CI 0.48–0.99) (P ⫽ 0.048). No significant differences in genotype or OR were observed between the THA groups for the remaining IL1 or IL6 SNPs (P ⬎ 0.05) (Table 3). RISK OF OSTEOLYSIS CONFERRED BY POLYMORPHISMS IN THA Table 3. Genotype distribution and minor allele carriage rates for the studied SNPs in subjects with osteolysis versus control subjects* Genotype frequency SNP ID no./site, alleles rs17561/IL1A ⫹4845 G/G G/T T/T rs4848306/IL1B ⫺3737 C/C C/T T/T rs16944/IL1B ⫺511 C/C C/T T/T rs1143634/IL1B ⫹3954 C/C C/T T/T rs419598/IL1RA ⫹2018 T/T T/C C/C rs1800797/IL6 ⫺597 G/G G/A A/A rs1800796/IL6 ⫺572 G/G G/C C/C rs1800795/IL6 ⫺174 G/G G/C C/C Minor allele carriage rate, % Control Osteolysis Control Osteolysis group group group group (n ⫽ 340) (n ⫽ 272) (n ⫽ 340) (n ⫽ 272) 159 144 36 136 110 22 53 49 93 172 75 69 139 63 73 75 164 141 34 128 110 31 52 52 184 137 18 159 94 16 46 41 180† 134 26 171 74 26 47‡ 37 125 149 65 90 119 60 63 67 308 30 0 240 30 0 9 11 122 164 54 89 135 47 64 67 * The genotype distributions within each group were in HardyWeinberg equilibrium (P ⬎ 0.05 by chi-square test), with the exception of the osteolysis group for the single-nucleotide polymorphism (SNP) rs419598 (P ⫽ 0.01 by chi-square test). † P ⫽ 0.007 versus osteolysis group, by chi-square test. ‡ P ⫽ 0.01 versus osteolysis group, by chi-square test. Haplotype analysis. Thirteen haplotypes (haplotype frequency range 32–1%) for the IL1 family SNPs and 6 haplotypes (haplotype frequency range 53–1.5%) for the IL6 SNPs were analyzed using Haploview 4.0. The least frequent IL6 haplotype (–174G/–572G/–597A) was associated with osteolysis (2.4% in the osteolysis group versus 0.8% in the control group; P ⫽ 0.02, calculated using Haploview software) (Table 4). No other IL6 or IL1 haplotypes were associated with osteolysis. PBMC stimulation study findings. Sixty subjects (mean ⫾ SD age 75 ⫾ 6 years, 30 men and 30 women) 3161 were recruited. The IL1RA ⫹2018 (rs419598) genotype distributions in this cohort were 33 with TT, 21 with CT, and 6 with CC (in Hardy-Weinberg equilibrium, P ⬎ 0.05). For both visit 1 and visit 2 PBMC stimulations, there was an allele–dose relationship between the number of copies of the C allele (0, 1, or 2) and increasing IL-1Ra mRNA expression (P ⫽ 0.023 by ANOVA at visit 1, P ⫽ 0.031 by ANOVA at visit 2, P ⫽ 0.033 by ANOVA for the combined data set) (Figure 1). Post hoc analysis within the repeated-measures ANOVA for the combined data set (Figure 1, bottom panel) gave a pooled mean ⫾ SEM estimate for mRNA expression (relative to ␤-actin) of 0.51 ⫾ 0.04 for the TT genotype, 0.57 ⫾ 0.05 for the CT genotype, and 0.81 ⫾ 0.10 for the CC genotype. IL-1Ra mRNA expression for the CC genotype was significantly greater than that for the TT genotype (P ⫽ 0.01 by ANOVA, after Bonferroni correction), but the significance of the difference between the CC and CT genotypes was borderline (P ⫽ 0.07 by ANOVA, after Bonferroni correction). An increase in IL-1Ra mRNA expression at 3 hours occurred following PBMC stimulation with all 3 agonists (LPS, Ti, and Ti plus LPS) compared with unstimulated PBMCs for the subjects at visit 1 (P ⬍ 0.01 for all comparisons, by post hoc analysis with Bonferroni correction). A similar pattern was replicated at the visit 2 PBMC stimulations (P ⬍ 0.01 for all comparisons). Revision status was not a predictor of IL-1Ra mRNA expression levels independent of rs419598 C allele copy number (n ⫽ 28 controls, n ⫽ 32 with previous revision; P ⬎ 0.05 by ANOVA for both visits). IL-1Ra mRNA expression for individual subjects was highly reproducible between stimulation studies (visit 1 and visit 2) for each stimulation condition. Pearson correlation coefficients (r) for mRNA expression between visit 1 and visit 2 for unstimulated cells and cells stimulated with LPS, Ti, or Ti plus LPS were 0.69, Table 4. IL6 haplotype frequencies in the osteolysis group versus the control group* Haplotype IL6 ⫺174/⫺572/⫺597 Haplotype frequency Haplotype frequency osteolysis group: control group P G/G/G C/G/A G/C/G G/C/A C/G/G G/G/A 0.528 0.391 0.028 0.019 0.016 0.015 0.50:0.55 0.40:0.39 0.03:0.03 0.02:0.02 0.018:0.014 0.024:0.008 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 ⬎0.05 0.02 * Data are not shown for IL1 family haplotypes (all P ⬎ 0.05). 3162 GORDON ET AL Genotype/phenotype mRNA expression analyses were not conducted for the IL6 haplotype –174G/ –572G/–597A that was associated with osteolysis in the case–control association study, since this haplotype was insufficiently frequent in the population to conduct PBMC stimulation assays. Genotype/phenotype mRNA expression analyses were not performed for IL1A and IL1B, since no significant associations between genotype and phenotype were identified in the case–control association study. DISCUSSION Figure 1. Effect of IL1RA ⫹2018 (rs419598) genotype on in vitro expression of mRNA for interleukin-1 receptor antagonist (IL-1Ra) in peripheral blood mononuclear cells obtained from 60 subjects who had undergone total hip arthroplasty. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the minimum and maximum values. Lines inside the boxes represent the median. Top, Results at subject visit 1; middle, results at subject visit 2; bottom, combined results of both visit data sets. The comparison variable is mRNA expression between the genotypes TT, CT, and CC across all conditions of stimulation, analyzed by repeated-measures analysis of variance (ANOVA). LPS ⫽ lipopolysaccharide; Ti ⫽ titanium. 0.64, 0.70, and 0.68, respectively (P ⬍ 0.001 for all correlations) (Figure 2). In this study we examined the association of SNPs in the IL1 gene family and in the IL6 gene with osteolysis after cemented THA. We found that the less common C allele at SNP rs419598 (IL1RA ⫹2018), which encodes the antiinflammatory cytokine IL-1Ra, was negatively associated with osteolysis. We also examined the relationship between alleles of SNP rs419598 and mRNA expression in stimulated PBMCs obtained from a clinically relevant population of patients who had undergone THA, comprising subjects without osteolysis and subjects who had previously expressed the osteolysis phenotype. Compared with the TT genotype, the CC genotype at rs419598 was associated with increased IL-1Ra mRNA production in response to PBMC stimulation. This effect was repeatable between subject attendances and robust to different stimulation conditions. A rare haplotype within the gene for IL-6 (–174G/–572G/–597A, with a frequency of ⬍5% in our population) was positively associated with osteolysis. However, this haplotype was insufficiently common to allow a PBMC stimulation study to be conducted. Our findings with respect to IL-6 are, however, consistent with a preliminary report by Kolundzic et al, who found that the individual SNPs at –572 and –597 were not associated with osteolysis (22), although they found that a compound genotype comprising –572CC and –597GA was associated with osteolysis. The SNP rs419598 (IL1RA ⫹2018) is associated with several chronic inflammatory conditions including Sjögren’s syndrome, alopecia areata, and atherosclerosis (30–32). It is also associated with pathologies of bone and joints, including osteoporotic fracture (33), and with susceptibility to RA (34,35). Our finding that alleles of this SNP are associated with differential mRNA expression does not, however, give evidence of a direct effect of the alleles on mRNA transcription or mRNA stability. This variant is a synonymous SNP in exon 2 of the IL1RA gene, and Clay et al have reported that this SNP has no effect on mRNA levels in the human keratinocyte RISK OF OSTEOLYSIS CONFERRED BY POLYMORPHISMS IN THA 3163 Figure 2. Pearson correlation coefficients comparing IL-1Ra mRNA production between visit 1 and visit 2 in peripheral blood mononuclear cells obtained from 60 subjects who had undergone total hip arthroplasty. All stimulations were performed independently at each visit. See Figure 1 for definitions. cell population (36). The C allele at this site is in 100% linkage disequilibrium with allele 2 of a variable-number tandem repeat (VNTR) in intron 2 of the IL1RA gene (36). The region surrounding the VNTR contains 3 putative protein binding sites—an interferon-␣ (IFN␣) silencer, an IFN␤ silencer, and an acute-phase response element—and this polymorphism may affect binding of transcription factors to these regulatory elements, or it may affect RNA stability (37). Several studies have investigated the relationship between the IL1RA VNTR or IL1RA ⫹2018 polymorphism and IL-1Ra mRNA or protein production. Wilkinson et al found that carriage of this allele resulted in an almost 2-fold increase in IL-1Ra mRNA induction in LPS-stimulated PBMCs compared with PBMCs carrying only the T allele at this site (38), and this is consistent with our study findings. Schrijver et al found an allele dose-response relationship between C allele copy number and IL-1Ra protein production in human whole blood stimulated with LPS (39). Danis et al have also described an association between this allele and increased IL-1Ra protein production by monocytes from healthy human subjects in response to granulocyte– macrophage colony-stimulating factor stimulation (40). However, this allele has also been associated in other studies with decreased mRNA or protein expression (41,42). These conflicting results may, however, reflect differences in mechanisms between the diseases studied, between pathologic and physiologic conditions, and between the in vivo and in vitro environment. Further functional studies in this area are warranted to define the potential role of this allelic variant on bone resorption activity in clinically relevant in vitro models of particle-induced osteolysis. Further, at an epidemiologic level, recent advances in medical imaging of osteolytic lesions using computed tomography and magnetic resonance imaging allow accurate volumetric measurement of osteolytic lesions in relation to implant wear (43,44). These techniques may be applied to evaluating the effect of identified allelic variants on the severity of osteolysis in prospective studies in vivo, and to quantitating the 3164 GORDON ET AL interaction between wear rate and genotype in the pathogenesis of osteolysis. In conclusion, we have shown that carriage of the IL1RA ⫹2018C allele appears to protect against osteolysis after cemented THA, and that this allele is positively associated with IL-1Ra mRNA production in response to clinically relevant stimuli, which may provide a mechanistic explanation for the observed association. At present, the functional role of the IL1RA ⫹2018 polymorphism in relation to the IL1RA intron 2 VNTR remains unclear. However, the finding that the ⫹2018C allele is overrepresented in patients with surviving THA implants compared with those with osteolysis supports the importance of the IL1 gene cluster family in osteolysis and aseptic loosening. AUTHOR CONTRIBUTIONS Dr. Wilkinson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Gordon, Kiss-Toth, Stockley, Eastell, Wilkinson. Acquisition of data. Gordon, Wilkinson. Analysis and interpretation of data. 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