Yasunobu Tamaki

Macrophages – Key Cells in the Response to Wear Debris from Joint Replacements

The generation of wear debris is an inevitable result of normal usage of joint replacements. Wear debris particles stimulate local and systemic biological reactions resulting in chronic inflammation, periprosthetic bone destruction, and eventually, implant loosening, and revision surgery. The latter may be indicated in up to 15% patients in the decade following the arthroplasty using conventional polyethylene. Macrophages play multiple roles in both inflammation and in maintaining tissue homeostasis. As sentinels of the innate immune system, they are central to the initiation of this inflammatory cascade, characterized by the release of proinflammatory and pro-osteoclastic factors. Similar to the response to pathogens, wear particles elicit a macrophage response, based on the unique properties of the cells belonging to this lineage, including sensing, chemotaxis, phagocytosis, and adaptive stimulation. The biological processes involved are complex, redundant, both local and systemic, and highly adaptive. Cells of the monocyte/macrophage lineage are implicated in this phenomenon, ultimately resulting in differentiation and activation of bone resorbing osteoclasts. Simultaneously, other distinct macrophage populations inhibit inflammation and protect the bone-implant interface from osteolysis. Here, the current knowledge about the physiology of monocyte/macrophage lineage cells is reviewed. In addition, the pattern and consequences of their interaction with wear debris and the recent developments in this field are presented.

Expression of Toll-like receptors and their signaling pathways in rheumatoid synovitis.

Objective. Toll-like receptors (TLR) recognizing endogenous and exogenous danger signals could play a role in rheumatoid arthritis (RA). Our aim was to describe the presence, localization, and extent of expression of TLR and their adapters. Methods. TLR 1, 2, 3, 4, 5, 6, and 9 receptors, and myeloid differentiation primary response protein 88, Toll/interleukin receptor (TIR) domain-containing adapter protein MyD88 adapter-like, and TIR domain-containing adapter-inducing interferon/TIR-containing adapter molecule-1 adapters were analyzed in RA (n = 10) and osteoarthritis (OA; n = 5) samples using real-time polymerase chain reaction (PCR). Their colocalization with cellular markers CD68, CD15, CD3, CD4, CD8, CD20, dendritic cell lysosomal-associated membrane protein (DC-LAMP), CD123, and 5B5 was analyzed in double immunofluorescence staining. Results. In RA, ß-actin standardized messenger RNA of TLR 2, 3, and 9 (p < 0.001) were particularly high. TLR 5 and 6 were also elevated (p < 0.05), but TLR 1 and 4 and adapters did not differ between RA and OA. In double-staining, TLR and adapters were strongly labeled in myeloid and plasmacytoid dendritic cells (DC), moderately in CD68+ type A lining cells/macrophages, and weakly to moderately in 5B5+ type B lining cells/fibroblasts. CD3+/CD4+ and CD3+/CD8+ T cells and CD20+ B cells in perivenular areas and in lymphoid follicles were moderately TLR- and weakly adapter-positive. In OA, TLR and adapters were weakly immunolabeled in vascular, lining, and inflammatory cells. Conclusion. RA synovium showed abundant expression of TLR. RA synovitis tissue seems to be responsive to TLR ligands. DC, type A cells/macrophages, and type B cells/fibroblasts are, in that order from highest to lowest, equipped with TLR, suggesting a hierarchical responsiveness. In RA, danger-associated molecular patterns to TLR interactions may particularly drive DC to autoinflammatory and autoimmune cascades/synovitis.

Increased Expression of Toll-like Receptors in Aseptic Loose Periprosthetic Tissues and Septic Synovial Membranes Around Total Hip Implants

Objective. Toll-like receptors (TLR) are transmembrane proteins found in various cells. They recognize infectious and endogenous threats, so-called danger signals, that evoke inflammation and assist adaptive immune reactions. It has been suggested that TLR play a role in periprosthetic tissues and arthritic synovium. Our objective was to elucidate tissue localization and functional roles of TLR in periprosthetic tissues in 2 different pathologic conditions, aseptic and septic implant loosening. Methods. For immunohistochemistry studies, aseptic synovial-like membranes of periprosthetic connective tissues (n = 15) and septic synovial capsular tissues (n = 5) were obtained at revision surgery and from salvage of infected totally replaced hips, respectively. Osteoarthritic synovial tissues were used for comparison (n = 5). Samples were processed for immunohistopathologic analyses for tissue colocalization of TLR with CD68 and/or CD15 using theAlexa fluorescent system. Total RNA was isolated from frozen tissues and converted into cDNA, TLR 2, 4, 5 and 9 sequences were amplified, and the products were quantified using real-time polymerase chain reaction. Results. Immunofluorescent staining showed colocalization of TLR 2, 4, 5, and 9 with CD68 in the focal monocyte/macrophage aggregates in aseptic synovial-like membranes from loose total hip replacements. TLR 2, 4, 5, and 9 were also found colocalized with CD15+ polymorphonuclear leukocytes and CD68+ mononuclear cells of the synovial membranes from septic total hip replacements. In osteoarthritic synovial tissues, expression of TLR was found only in vascular cells and mononuclear cells, and the reactivity was weak. mRNA levels of TLR 2, 4, 5, and 9 were increased in both aseptic and septic periprosthetic tissues. TLR 2 and 5 were significantly higher than TLR 4 and 9 in aseptic and septic samples. Conclusion. Peri-implant tissues were well equipped with TLR in both aseptic and septic conditions. TLR 2- and TLR 5-mediated responses seemed to dominate. In aseptic loosening, monocytes/ macrophages were the main TLR-equipped cells apparently responsible for alarmin-induced responses. This could lead to production of inflammatory cytokines and extracellular matrix-degrading proteinases after phagocytosis of wear debris derived from an implant, but in septic cases they eventually respond to microbial components. Thus, inflammatory cells in both aseptic and septic tissues were equipped with TLR, providing them with responsiveness to both endogenous and exogenous TLR ligands.

Toll-like receptors and their adaptors are regulated in macrophages after phagocytosis of lipopolysaccharide-coated titanium particles.

Macrophages phagocytose metallic wear particles and produce mediators, which can induce cellular host response and aseptic implant loosening. Lipopolysaccharide (LPS) on the wear debris can stimulate macrophages via Toll‐like receptor 4 (TLR4) and enhance the response. However, the precise functional role and interaction of TLRs and their adaptor molecules is still unclear. Rat bone marrow macrophages were stimulated with titanium particle (Ti) coated by LPS (Ti/LPS+) and LPS‐free Ti (Ti/LPS−). mRNA levels of cytokines, TLRs and their adaptor molecules were measured using real time PCR. mRNA levels of TNF‐α, IL‐1β, and IL‐6 increased in Ti/LPS+ than Ti/LPS−. In contrast, mRNA levels of TLR4, TLR5, and TLR9 decreased in Ti/LPS+ compared to Ti/LPS−. mRNA levels of MyD88, IRAK1, IRAK4 decreased gradually, and TRAF6 underwent an initial transient increase, followed by suppression in Ti/LPS+. However, mRNA levels of TLR2 and IRAK2 increased after phagocytosis of Ti/LPS+ than Ti/LPS−. The increased expressions of proinflammatory cytokines found in Ti/LPS+ indicated that their productions cytokines could be enhanced by phagocytosis of LPS‐coated particles. Subsequent down‐regulation of TLR4, TLR5, TLR9, MyD88, IRAK1, and IRAK4 suggests that self‐protective mechanisms to regulate excessive host responses are activated in macrophages. Increase of TLR2 and IRAK2 and a transient increase of TRAF6 in Ti/LPS+ suggest that another possible pathway to modulate TLR‐mediated cellular response to prolong inflammatory response in foreign body reaction of aseptic loosening. This down‐ and/or up‐regulation of the potential TLR‐mediated responses to LPS‐coated particles reflects the proactive behavior of effector cells.

Profile of toll-like receptor-positive cells in septic and aseptic loosening of total hip arthroplasty implants

Distinction between the two major complications of total hip replacement surgery, septic bacterial culture-positive and aseptic bacterial culture-negative osteolysis and loosening, is difficult due to the eventual role of bacterial remnants and biofilms, which are recognized by cells provided by toll-like receptors (TLRs) of the innate immune system. It was hypothesized that cell typing and TLR mapping might provide new information on the pathomechanisms of loosening. To test this hypothesis, septic (n = 10) and aseptic (n = 5) interface tissue as well as mildly inflamed osteoarthritic synovial membrane (n = 5) samples were characterized and compared using antibodies against several cell line-specific markers, including fibroblast, monocyte/macrophage, T cell, B cell, plasma cell and neutrophil markers, and TLRs. In osteoarthritic synovium, TLR-positive cells were restricted to surface tissues and only few inflammatory cells were detected, whereas aseptic interface was heavily infiltrated with monocyte/macrophages, which were also the major TLR-positive cell type rendering the tissue reactive to TLR ligands. Interestingly, septic cases contained also neutrophil and lymphocyte infiltrates of which especially B-cell infiltrates might be clinically useful in discriminating the two major complications of the joint replacement surgery. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Modulation of mouse macrophage polarization in vitro using IL-4 delivery by osmotic pumps.

Modulation of macrophage polarization is emerging as promising means to mitigate wear particle-induced inflammation and periprosthetic osteolysis. As a model for continuous local drug delivery, we used miniature osmotic pumps to deliver IL-4 in order to modulate macrophage polarization in vitro from nonactivated M0 and inflammatory M1 phenotypes towards a tissue regenerative M2 phenotype. Pumps delivered IL-4 into vials containing mouse bone marrow macrophage (mBMM) media. This conditioned media (CM) was collected at seven day intervals up to four weeks (week 1 to week 4 samples). IL-4 concentration in the CM was determined by ELISA and its biological activity was assayed by exposing M0 and M1 mBMMs to week 1 or week 4 CM. The IL-4 concentration in the CM approximated the mathematically calculated amount, and its biological activity was well retained, as both M0 and M1 macrophages exposed to either the week 1 or week 4 CM assumed M2-like phenotype as determined by qRT-PCR, ELISA, and immunocytochemistry. The results show that IL-4 can be delivered using osmotic pumps and that IL-4 delivered can modulate macrophage phenotype. Results build a foundation for in vivo studies using our previously validated animal models and provide possible strategies to locally mitigate wear particle-induced macrophage activation and periprosthetic osteolysis.

Lipoteichoic acid modulates inflammatory response in macrophages after phagocytosis of titanium particles through Toll-like receptor 2 cascade and inflammasomes

Toll-like receptor 2 (TLR2) and nucleotide-binding and oligomerization domain-like receptors with a pyrin domain 3 (NLRP3) inflammasomes have been presumed to participate in the pathogenesis of aseptic implant loosening. The aim of this study is to analyze the cellular localization of TLR2 and NLRP3 inflammasomes in the periprosthetic tissue from aseptically loose hip implants as well as the expression of these molecules in macrophages stimulated in vitro with titanium particles (Ti) coated with lipoteichoic acid (LTA). Using immunohistochemistry, immunoreactivity of TLR2 and NLRP3 inflammasomes was found in macrophages within the foreign body granulomatosis. Using RAW264.7 cells, stimulation with Ti increased the messenger RNA (mRNA) levels of TLR2 and TNF-α. Stimulation with LTA-coated Ti enhanced mRNA levels of NLRP3 and IL-1β, whereas reinforced secretion of IL-1β was not detected in spite of marked release of TNF-α. Finally, the same cells with silenced Irak2, an adaptor protein in the TLR2 cascade, suppressed this NLRP3 upregulation. This study suggests that TLR2 and NLRP3 inflammasomes are factors involved in cross-talk mediating the foreign body type response to wear particles. In addition, discrepant behavior in the release between TNF-α and IL-1β release may explain the variable pathomechanisms of aseptic implant loosening without acute inflammatory reactions.

Edoxaban for prevention of venous thromboembolism after major orthopedic surgery

Correspondence: Hiroyuki Kawaji Department of Orthopaedic Surgery, Saiseikai Yamagata Saisei Hospital, 79-1 Okimachi, Yamagata, Japan Tel +812 3682 1111 Fax +812 3682 0122 Email hiroyuki.kawaji@ameria.org Abstract: Fatal pulmonary thromboembolism is the most serious complication following surgery. Patients undergoing major orthopedic surgeries, including total hip replacement, total knee replacement, and hip fracture surgery, represent a group at particularly high risk of venous thromboembolism. Therefore, prophylaxis for thromboembolic events has been of great concern to surgeons. Edoxaban is a novel, orally available, and highly specific and direct factor Xa inhibitor. This new agent was approved for the prevention of venous thromboembolism in patients undergoing major orthopedic surgery, including total hip replacement, total knee replacement, and hip fracture surgery, by the Japanese Ministry of Health, Labor, and Welfare in 2011. Preclinical and Phase I clinical trials demonstrated several promising properties. Its rapid absorption and short life-time in blood are known. Edoxaban inhibits factor Xa activity directly and selectively. It also has a strong antithrombotic effect without any influence of food intake. Coagulation monitoring is not required. Edoxaban has predictable linear pharmacokinetic and pharmacodynamic profiles. Phase II and III clinical trials have been completed to examine its efficacy and safety in patients undergoing major orthopedic surgery. In these clinical trials, oral administration of edoxaban showed efficacy superior to that of oral placebo or subcutaneously administered dalteparin or enoxaparin. Edoxaban can be regarded as a first choice to prevent venous thromboembolism after major orthopedic surgery.

NF-κB decoy oligodeoxynucleotide inhibits wear particle-induced inflammation in a murine calvarial model

Wear particles induce periprosthetic inflammation and osteolysis through activation of nuclear factor kappa B (NF-κB), which up-regulates the downstream target gene expression for proinflammatory cytokines in macrophages. It was hypothesized that direct suppression of NF-κB activity in the early phases of this disorder could be a therapeutic strategy for preventing the inflammatory response to wear particles, potentially mitigating osteolysis. NF-κB activity can be suppressed via competitive binding with double stranded NF-κB decoy oligodeoxynucleotides (ODNs) that blocks this transcription factor from binding to the promoter regions of targeted genes. In this murine calvarial study, clinically relevant polyethylene particles (PEs) with/without ODN were subcutaneously injected over the calvarial bone. In the presence of PE particles, macrophages migrated to the inflammatory site and induced tumor necrosis factor alpha (TNF-α) and receptor activator of nuclear factor kappa B ligand (RANKL) expression, resulting in an increase in the number of osteoclasts. Local injections of ODN mitigated the expression of TNF-α, RANKL, and induced the expression of two anti-inflammatory, antiresorptive cytokines: interleukin-1 receptor antagonist and osteoprotegerin. Local intervention with NF-κB decoy ODN in early cases of particle-induced inflammation in which the prosthesis is still salvageable may potentially preserve periprosthetic bone stock.

Distribution of podoplanin in synovial tissues in rheumatoid arthritis patients using biologic or conventional disease-modifying anti-rheumatic drugs.

OBJECTIVE Podoplanin (PDPN) mediates tumor cell migration and invasion, which phenomena might also play a role in severe rheumatoid arthritis (RA). Therefore, the precise cellular distribution of PDPN and its relationships with inflammation was studied in RA treated with biologic disease-modifying anti-rheumatic drugs (DMARD) or conventional DMARDs (cDMARD). METHODS PDPN+ cells were immunostained by NZ-1 mAb, and scored (3+; >50%/ area, 2+; 20%- 50%, 1+; 5%-20%, 0: <5%) in synovial tissues from RA treated with biologic DMARDs (BIO, n=20) or cDMARD (n=20) for comparison with osteoarthritis (OA, n=5), followed by cell grading of inflammation and cell-typing. RESULTS Inflammatory synovitis score was 1.4 in both BIO and cDMARD, compared to only 0.2 in OA. PDPN+ cells were found in the lining layer (BIO 1.6, cDMARD 1.3, OA 0.2) and lymphoid aggregates (BIO 0.6, cDMRD 0.7, OA 0.2), and correlated with RA-inflammation in BIO- and cDMARD-groups in both area (r=0.7/0.9, r=0.6/0.7, respectively p<0.05). PDPN was expressed in CD68+ type A macrophage-like and 5B5+ type B fibroblast-like cells in the lining layer, and in IL- 17+ cells in lymphoid aggregates in RA. CONCLUSION PDPN was markedly increased in the immunologically inflamed RA synovitis, which was surgically treated due to BIO- and cDMARD-resistant RA. PDPN may have potential of a new marker of residual arthritis in local joints for inflammation-associated severe RA.