Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles
Introduction
Aseptic loosening of total joint replacements is the most common cause of revision surgery [1]. One potential contributing factor is implant wear resulting in the release of numerous particles into the joint space [2]. Macrophages phagocytose the wear debris and synthesize cytokines such as tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), IL-6 and others, which, among other actions, lead to osteoclast activation and breakdown of the surrounding bone, compromising the long-term outcome of the prosthesis [3], [4]. Although it is widely accepted that aseptic loosening of joint replacements is largely driven by wear particles [4], the mechanism by which particles are recognized and activate macrophages remains largely undetermined.
Binding of pathogens to macrophages can induce responses of the innate immune system and lead to the induction of adaptive immunity [5]. The Toll-Like Receptors (TLR) are a group of evolutionarily conserved transmembrane receptors that function as signaling receptors. Each member of the TLR family is activated by a different stimulus and subsequently activates a response to that type of pathogen [6]. For example, TLR-4 signals the presence of lipopolysaccharide (LPS) on the cell membrane of gram-negative bacteria and activates an inflammatory response [7]. When the majority of TLR’s are stimulated, they interact with an adapter protein called Myeloid Differentiation primary response gene 88 (MyD88), which couples the TLR to downstream signaling kinases, eventually culminating in the activation (by translocation from the cytoplasm to the nucleus) of the transcription factor nuclear factor κB (NF-κB) [8] (Supplementary Fig. 1). However, there exist MyD88-independent signaling pathways for TLR3 and TLR4. TLR4 possesses two options for signal transduction, either to signal through MyD88 or through TIR domain containing adapter inducing interferon-beta (TRIF), while TLR3 can signal only through TRIF [6]. Although the MyD88-dependent and MyD88-independent pathways utilize distinct adapter proteins, both signaling pathways involve the activation and nuclear translocation of NF-κB which will lead to the expression of numerous proinflammatory cytokines. Osteoclastogenesis is also related to activation of the NF-κB pathway, and previous studies have demonstrated that wear debris-induced inflammatory osteoclastogenesis occurs in part by an increase in cytokine production and osteoclast differentiation, both of which involve the NF-κB pathway [9], [10].
Considering the relationship between TLR signaling, NF-κB signaling and the known proinflammatory cytokines associated with aseptic loosening of joint implants, we proposed that TLRs play a critical role in the recognition of wear particles.
Section snippets
Preparation of PMMA particles
PMMA particles (Polysciences) 1–10 μm in diameter, were washed five times with 70% ethanol and incubated overnight with shaking at 4 °C. The particles were then washed extensively with phosphate-buffered saline (PBS) and resuspended to make a concentrated 16% v/v stock solution. The particles were free of endotoxin using a high-sensitivity Limulus amebocyte lysate assay (BioWhittaker).
Quantification of TNF-α release
Macrophages were exposed to PMMA particles and samples from the culture media were collected at the indicated
Dose-dependent relationship between PMMA particle concentration and TNF-α
To investigate whether the TLR pathway contributes to the recognition of PMMA particles we identified an optimal particle concentration which stimulates robust production of TNF-α by the phagocytic cells. To simulate the in vivo scenario of macrophages being exposed to orthopedic implant wear-debris particles, the macrophage cell line RAW 264.7 was incubated in vitro with PMMA particles. At 4, 12 and 24 h post-particle exposure we observed a linear correlation between PMMA particle dose and
Discussion
The aim of this study was to investigate whether the inflammatory response induced by prosthetic implant wear-debris particles is mediated by components of the TLR signaling pathway. To address this question we first challenged a murine macrophage cell line with PMMA particles and compared the expression of TNF-α. Macrophages treated with a small peptide inhibitor of MyD88 demonstrated significantly diminished production of TNF-α compared to untreated control macrophages. We next tested whether
Conclusions
This study demonstrates that the response to PMMA particles is dependent in part on MyD88, as part of the TLR signaling pathway. This conclusion is supported by evidence demonstrating that in both a murine macrophage cell line and primary mouse macrophages, disruption of MyD88 signaling reduces PMMA particle-induced production of TNF-α. In contrast, the inflammatory response to PMMA particles by TRIF−/− BMDM was enhanced, possibly because of a compensatory increase in the expression of numerous
Acknowledgments
We thank Dr. Judith Hellman (University of California San Francisco) for generously providing us with the wild type, MyD88 knockout and TRIF knockout bone marrow derived macrophages. This work was supported in part by the Robert L and Mary Ellenburg Chair in Surgery (S.B.G), the Stanford Medical Scholars Program (J.I.P) and the Orthopaedic Research and Education Foundation (J.I.P).
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