Abstract

Background

Heterotopic ossification (HO) is lamellar bone formation in the soft tissues following trauma or joint replacement for osteoarthritis (OA). A genome wide association study of HO patients after total hip arthroplasty for OA has identified Kinesin Family Member 26B (KIF26B) as a gene associated with HO severity. KIF26B has previously been associated with HO in mice.

Hypothesis and aims: We hypothesised that Kif26b regulates the osteogenic trans-differentiation of myoblasts; a possible mechanism of HO. Using an in vitro model, we wished to establish whether Kif26b is involved in HO formation and to explore the molecular mechanism.

Total hip arthroplasty (THA) wear debris induced macrophage expression of pro-inflammatory cytokines has been associated with osteolysis both in vitro and in animal and human subjects. Interleukin-1 receptor antagonist (IL-1RA) is an anti-inflammatory cytokine which may limit bone destruction. Polymorphisms (SNPs) within the IL-1RN gene are associated with differences in susceptibility to infectious and inflammatory conditions and disorders of bone remodelling. This study investigated the association between the IL-1RA+2018T/C SNP (rs419598) and osteolysis after THA, and with mRNA and protein expression in an in-vitro wear debris-macrophage stimulation assay.

611 North European Caucasians who had received a cemented THA for primary osteoarthritis were genotyped for the IL-1RN+2018 SNP using Taqman methods. 62 subjects with a Charnley THA were selected from the genotyping population. Control subjects had no radiographic osteolysis and the osteolysis group had previously undergone revision surgery for aseptic loosening. Peripheral blood mononuclear cells were extracted and stimulated with endotoxin-stripped titanium particles (TiCL, endotoxin level 0 Eu/ml) and endotoxin-stripped particles with adherent LPS added back (TiAB, endotoxin level 140 Eu/ml); non-stimulated and LPS-stimulated cells were used as negative and positive controls. Cell lysate IL-1RA mRNA levels were assessed by rqRT-PCT following a 3-hour stimulation. Cell supernatant IL-1RA protein levels were assayed after 24 hours stimulation using a multiplex method.

The IL-1RN+2018C allele was underrepresented in patients with osteolysis after THA versus control THA subjects (chi-squared test 5.96, P=0.015). After correction for other risk factors for osteolysis, the adjusted odds ratio for osteolysis associated with carriage of the IL-1RN+2018C SNP was 0.69 (0.48 to 0.99, p=0.048). IL-1RA mRNA expression increased linearly with IL-1RN+2018C allele copy number in gene-dose dependent manner (ANOVA p=0.013). The IL-1RA+2018C allele did not significantly affect IL-1RA protein expression (ANOVA p> 0.05), however a similar trend towards increased levels with increased C allele copy number was observed.

Carriage of the IL-1RA+2018C allele is associated with both a decreased risk of osteolysis after THA and increased IL-1RA mRNA expression in-vitro. The mechanism for this functional effect remains unclear, however these findings support the importance of the IL-1RA in osteolysis and aseptic loosening.

Introduction: Immune responses in patients susceptible to aseptic loosening may differ to those without this susceptibility. We compared stimulated cytokine mRNA and protein expression in peripheral blood mononuclear cells (PBMC) in 34 subjects (M:F 16:18; mean age 75 years) with previous revision surgery for aseptic loosening versus 28 subjects (14:14; 75 years) with well-fixed implants after Charnley THA for osteoarthritis.

Methods: Extracted PBMCs were stimulated with endotoxin (LPS 200ng/mL), endotoxin-free titanium particles (Ti, endotoxin level =0 Eu/mL), or particles with adherent LPS (TiLPS, 140 Eu/mL). Cell lysate IL-1α, IL-1β, IL-1RA, IL-6, IL-10, IL-18, and TNF mRNA were assayed after 3 hours stimulation using standard rqRT-PCR techniques. Cell supernatant IL-1β, IL-1RA, IL-6 and TNF protein were assayed after 24 hours stimulation using a multiplex method.

Results: mRNA and protein levels in non-stimulated cells were lower in revision versus control subjects for all cytokines (p< 0.05 all analyses). mRNA expression relative to baseline was greater in revision subjects versus controls for all cytokines and all modes of stimulation (LPS, Ti, and TiLPS, p< 0.05 all analyses). LPS induced the greatest inflammatory cytokine response at both the mRNA and protein level in both groups, TiLPS particles induced a more attenuated response, and responses to Ti particles were weakest. In the control group endotoxin free particles showed a negative cytokine mRNA response for IL-1α, IL-1β, and IL-6 (p< 0.05), and reduced protein levels for IL-1β, IL-1RA, IL-6, and TNF versus non-stimulated cells (p< 0.05).

Discussion: Patients with a susceptibility to aseptic loosening have lower baseline but greater stimulated immune responses versus patients without loosening that may contribute to the pathogenesis of aseptic loosening.

Phagocytosis of wear particles by perimplant macrophages results in cytokine release and osteoclast activation and osteolysis. Some investigators have proposed that this response may be mediated by adherent endotoxin. The aim of this study was to determine the role of endotoxin in modulating pro-inflammatory cytokine mRNA expression of macrophages when stimulated with titanium particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR)

Human peripheral blood mononuclear cells were isolated from healthy subjects and plated in chamber slides. Three types of titanium particles were prepared; commercially pure titanium particles (cpTi), endotoxin stripped particles and endotoxin stripped particles with endotoxin (LPS) added back. Endotoxin levels of 450, 0 and 140 Eu/ml respectively were confirmed by high sensitivity Limulus Amebocyte Lysate assay. Macrophages were stimulated with particle concentrations of 0, 8.3, 83 and 830 particles per cell at time points 0 and 3 hours. LPS (200ng/ml) was used as a positive control. rqRT-PCR was performed using standard techniques.

Stimulation of human macrophages with cpTi demonstrated a significant dose dependent increase in TNFα, IL-1A, IL-1B and, IL-6. (Kruskal-Wallis p=0.01, p=0.017, p=0.001 and p=0.013 respectively). IL-18 mRNA levels were not increased (P> 0.05). The expression of mRNA following stimulation with the highest dose of titanium particles was similar to that following LPS stimulation. Endotoxin-free cpTi particles did not elicit any increase in mRNA expression above base line levels (P > 0.05, all cytokines). This lack of response was rescued in endotoxin-stripped particles with LPS added back. Particle dose dependent increases in cytokine mRNA levels were observed for TNFα, IL-1A, IL-1B and, IL-6 mRNA but not IL-18 (p=0.01, p=0.01, p=0.01, p=0.05 and p=0.> 0.05 respectively).

Our results show that adherent endotoxin plays a role in modulating particle induced pro-inflammatory cytokine mRNA expression in-vitro. Further study is required in evaluating the role of adherent endotoxin in vivo

Cytokine mediated activation of osteoclasts can lead to peri-implant osteolysis and aseptic loosening. The aim of this study was to determine the IL-1β and TNFα mRNA cytokine expression profile of human macrophages when stimulated with polyethylene particles using relative quantitative real-time polymerase chain reaction (rqRT-PCR).

Human peripheral blood monocytes or human monocytes from the cell line THP-1 were used in this study. rqRT-PCR conditions were optimized by stimulating human macrophages with 200ng/ml lipopolysaccharide (LPS). The median CV% value for duplicate measures was 12.6 (range 4.5–54). Stimulation assays were performed using unfractionated endotoxin-free commercial polyethylene particles (median size 7μm); or fractionated particles (size range 0.1–1.2μm). Human macrophages were stimulated with high dose unfractionated polyethylene particles at 0, 3500 or 10500 mm³/cell or with fractionated polyethylene particles at 0 and 100mm³/cell at time points 0 and 3 hours. Low dose unfractionated polyethylene stimulation was performed on THP-1 cells at 0, 50, 100, 1000 and 10000 mm³/cell. In all experiments LPS stimulation was used as a positive control. RNA was extracted and rqRT-PCR was performed using standard techniques

High dose unfractionated polyethylene stimulation did not result in a significant difference in cytokine mRNA levels between groups. Using fractionated polyethylene, a small increase in IL-1β mRNA was identified (21% versus maximal stimulation using LPS). Low dose unfractionated polyethylene stimulation of THP-1 cells demonstrated dose dependent decreases in TNFα and IL-1β mRNA expression that was not due to inhibition of RNA extraction or a decrease of cell viability.

Endotoxin-free polyethylene particles do not appear to be a major stimulus for IL-1β and TNFα mRNA production as measured by rqRT-PCR. We did observe a small positive effect on IL-1β mRNA expression using a fractionated polyethylene stimulus. However it remains unclear whether this effect is due to fractionation of particles into the submicron range or is due to introduction of endotoxin during the filtration process.

Polyethylene wear particle-induced osteolysis is a major cause of implant failure after total hip arthroplasty (THA). Tumour necrosis factor (TNF) is a pro-inflammatory cytokine that is thought to play a pivotal role in this process. We have recently shown that carriage of the −238 ‘A’ allele in the TNF gene promoter is associated with a higher rate of osteolysis after THA versus carriage of the [more common] ‘G’ allele. The aim of this study was to determine the effect of this polymorphism on TNF gene transcriptional activation in response to polyethylene particle stimulation using a luciferase reporter gene assay.

A 691 bp fragment (−585 to +106) of the TNF gene was amplified by polymerase chain reaction and directionally cloned into the PGL3.basic vector (Promega, Madison, WI). Insert sequences were checked using an ABI 377 DNA sequencer (PE Applied Biosystems, Foster City, CA). RAW264.7 murine macrophage-like cells in rapid growth phase were transfected with plasmids containing either the TNF-238G allele or the TNF-238A allele. pTK-RL (Promega), that expresses the Renilla luciferase gene under the control of Herpes simplex virus minimal promoter, was used as a transfection control. The cells were then either left unstimulated or were induced using polyethylene particles generated from a hip simulator. Lipopolysaccharide (LPS) and LTA (Lipoteichoic acid) were used as positive controls. Luciferase reporter activity was measured after 4 hours (Dual luciferase assay, Promega Corp., Southampton, U.K.) and the relative firefly luciferase activity was calculated. Results were analysed using repeated measures ANOVA.

Polyethylene particle stimulation at concentrations of 0, 1, 15, and 30mg/mL resulted in relative luciferase activities (mean (SD)) of 21.4 (2.9), 36.2 (8.2), 45.9 (11.1), and 40.7 (5.1) for the −238A allele; and 19.7 (5.0), 26.4 (8.0), 35.9 (2.3), and 32.4 (2.4) for the −238G allele (ANOVA P=0.01). LPS and LTA stimulation also resulted in increased reporter activity for −238A versus −238G (ANOVA P=0.02 and P=0.04, respectively).

The promoter allele TNF-238A results in higher levels of transcriptional activation versus the TNF-238G allele in response to a clinically relevant stimulus, and provides functional evidence for the significance of this polymorphism in the development of osteolysis after THA.