Tomoyuki Hirayama

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.

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.

Outcome of Nonoperative Treatment for Humeral Medial Epicondylar Fragmentation Before Epiphyseal Closure in Young Baseball Players

Background: Nonoperative treatment for humeral medial epicondylar fragmentation in baseball players, involving prohibition and limitation of throwing, has been reported to give good results. However, in some cases, such nonoperative treatment fails to yield an acceptable outcome. Hypothesis: In nonoperative treatment for patients with medial epicondylar fragmentation, achievement of bone union of the fragmentation provides better clinical outcomes compared with those of patients with delayed bone union or nonunion. Study Design: Cohort study; Level of evidence, 3. Methods: Fifty-five young baseball players with medial epicondylar fragmentation before epiphyseal closure, aged between 9 and 13 years (mean, 11.0 years), participated in this study. They belonged to baseball teams in a youth league and underwent nonoperative treatment involving prohibition of throwing for an average of 2.0 months and subsequent limitation of throwing for an average of 1.8 months. We investigated whether achievement of bone union of the fragmentation was associated with better clinical outcomes. Results: Bone union was achieved in 40 (73%) of 55 participants at 6 months after initial presentation, 31 (76%) of 41 participants at 1 year, and 32 (94%) of 34 participants at 2 years. Elbow pain was present in 7 participants (17%) at 1 year after initial presentation and in 6 participants (18%) at 2 years. At 1 year after initial presentation, statistical analysis showed that most participants with elbow pain had significant fragmentation (P = .0055). At 2 years after initial presentation, there was no significant relationship between elbow pain and medial epicondylar fragmentation (P = .32). Statistical analysis also showed that, at both 6 months and 1 year after initial presentation, bone union was significantly delayed in most participants who had not accepted nonoperative treatment and consequently resumed throwing vigorously before bone union. Conclusion: At 1 year after initial presentation, bone union of the medial epicondylar fragmentation was correlated with a decreased prevalence of elbow pain. At 6 months and 1 year after initial presentation, delayed bone union of the medial epicondylar fragmentation was associated with resumption of throwing at maximum strength before bone union had occurred.

Inflammatory immune cell responses and Toll-like receptor expression in synovial tissues in rheumatoid arthritis patients treated with biologics or DMARDs

Biologic antirheumatic drugs (BIO) have been reported to be potent therapeutic agents in the prevention of inflammatory joint destruction in rheumatoid arthritis (RA). The aim of this study was to investigate the immune-inflammatory cells, including Toll-like receptor (TLR)-equipped cells, in synovial tissue samples from RA patients on BIO compared to patients, who are only on conventional disease-modifying antirheumatic drug (DMARD). We analyzed immune-inflammatory cells in RA synovitis in patients of BIO group (n = 20) or DMARD group (n = 20). The grading scores of synovitis was 1.7 and 1.8 in each BIO and DMARD group and correlated best with the CD3+ T (r = 0.71/0.70, p < 0.05) and CD20+ B (r = 0.80/0.84, p < 0.05) cells in the both groups, but less well with the CD68+ macrophages and S-100+ dendritic cells (DCs). Interestingly, both T (116 vs. 242, p < 0.05) and B (80 vs. 142, p < 0.05) cell counts were lower in the BIO than in the DMARD group, whereas macrophage and DC counts did not differ. In contrast, the C-reactive protein (CRP) and disease activity score DAS28-CRP did not show clear-cut correlations with the inflammatory grade of the synovitis (r range, 0–0.35). Similar numbers of cells immunoreactive for TLR-1 to TLR-6 and TLR-9 were found in synovitis in both groups. Patients clinically responding to biologics might still have the potential of moderate/severe local joint inflammation, composed in particular of and possibly driven by the autoinflammatory TLR+ cells.

AB0408 Are Atypical Femoral Fractures in Rheumatic Patients Increasing

Background Atypical femoral fractures with low-energy or lack of trauma have been reported to relate using of the bisphosphonates (BPs) and glucocorticoids for a long time, affecting collagen diseases1,2. Objectives The aim of this study was to analyze the atypical femoral fractures in rheumatic patients in our four institutes retrospectively. Methods We investigated the cases of atypical femoral fractures summarized by the American Society for Bone and Mineral Research (ASBMR) Task Force 20101 among our out-rheumatic patients from 2009 to 2014. Results We have 1,091 out-rheumatic patients/year in our four institutes from 2009 to 2014. The patients with atypical femoral fractures were 8 limbs in six women (0.12%) in six years. Three limbs were injured at 2013, and five at 2014, including two cases has both side atypical femoral fractures (Fig. 1). The mean age of them was 51 year-old (38-73). As comorbid conditions, two patients has dermatomyositis, systematic lupus erythematosus, one rheumatoid arthritis and one polyarteritis nodosa. Fracture types were seven subtrochanteric fractures and one diaphyseal femoral fracture. All patients received BPs and prednisolone (PSL). Mean duration of receiving the drugs was 75 months (36-120) and 114 months (60-180), respectively. Mean dosage of PSL was 15 mg/day (5-30). After affecting the fractures, BPs were quitted and the surgery using intramedullary nail fixation were performed in all cases. One case had the surgical-site infection. Teriparatide was induced excepted one case and therapy of low-intensity pulsed ultrasound was started for all cases after healing their operated wounds. Mean duration of post-operative observation was 12 months (5-23). At the latest follow-up, five femurs were observed the sign of union at fracture site on X-ray or computed tomography of their femurs, but not other three femurs. Mean duration of union of the fracture site was 10 months (6-13) in five femurs. Conclusions Eight atypical femoral fractures were observed in 2013-14, but not in 2009-2012. Atypical femoral fracture may increase year by year. The careful management and treatment for the atypical femoral fractures in rheumatic patients were required even after the surgery, because our all cases have been observed the delayed union or non-union of fracture site at their latest follow-up3. References Shane E, et al. JMBR, 2010. Blacks DM, et al. NEJM, 2010. Thompson RN, et al. JBJS 2012. Disclosure of Interest None declared

AB0090 Is Podoplanin A New Candidate of Inflammatory Markers for Rheumatoid Arthritis

Background Podoplanin, known as platelet aggregation-inducing factor in the haematogenous metastasis of tumor cells [1], is a small, 38- to 40-kDa, mucin-type transmembrane glycoprotein normally expressed on human lymphatic endothelia, basal epithelial keratinocytes, alveolar type I, cancer cells [2] and possible potent molecule of inflammation. The expression of podplanin on inflammatory cells, such as fibroblast-like synoviocytes, follicular dendritic cells and Th17 T cells, is up-regulated when stimulated by pro-inflammatory cytokines [3,4]. Objectives The aim of this study was investigated to clarify its tissue and cellular distribution in the rheumatoid synovium. Methods 1) Samples; The synovial tissue samples were obtained surgically from the patients with rheumatoid arthritis (RA) treated with biologic disease-modifying anti-rheumatic drug (DMARD) (BIO, n=20) or conventional DMARD (cDMARD, n=20) and osteoarthritis (OA, n=5). 2) Immunohistochemistry; Tissue samples were fixed in 4% paraformaldehyde and embedded in Tissue-Tek OCT compound and snap-frozen. Serial 5-μm-thick frozen tissue sections were cut and stained by rat monoclonal anti-human podoplanin (NZ-1) using the avidin–biotin–peroxidase complex (ABC) method. 3) Double-labeling; The sections were incubated with the mouse monoclonal antibody, anti-CD68, anti-fibroblast 5B5, and rabbit polyclonal anti-IL17 with the rat monoclonal anti-human podoplanin. The sections were then incubated with secondary antibodies, Alexa Fluor® 488 or 568 before mounting. 4) Scoring; Podoplanin+ cells were scored (3+; >50%/area, 2+; 20%>50%, 1+; 5%>20%, 0: <5%) in the rheumatoid synoial tissues with analyses of inflammatory grading (0-3) and cell-typing. 5) Statistical analysis; Mann–Whitney U test and Spearmans rank correlation coefficient analysis were performed using the PASW 18 software. Values of p<0.05 were considered to indicate statistical significance. Results Inflammatory grading score was 1.4 in both BIO and cDMARD, and 0.2 in OA. Podoplanin+ cells were expressed in the lining layer (BIO 1.6, cDMARD 1.3, OA 0.2) (Fig. 1) and lymphoid aggregation (BIO 0.6, cDMRD 0.7, OA 0.2), which correlated with grading of RA synovium in both BIO and cDMARD (r=0.7/0.9, p<0.05), not OA (r=0.2). Podoplanin was markedly expressed in CD68+ type A macrophages-like and 5B5+ type B fibroblast-like cells in the lining layer and IL-17+ cells in lymphoid aggregations of RA. Figure 1 Conclusions Podoplanin was markedly expressed in the immunologically inflamed synovium and correlated to inflammatory, which was surgically treated due to progressive arthritis against both BIO and cDMARD. It indicates that podplanin will be a possible new therapeutic target in the treatment of RA. References Kato Y, et al: J Biol Chem 2003; 278: 51599–51605. Martín-Villar E, et al: Int J Cancer 2005; 113: 899-910. Ekwall AK, et al: Arthritis Res Ther 2011; 13: R40. Miyamoto Y, et al: Mol Immunol 2013; 54:199-207. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3532

AB0091 The Concentration of Serum Interleukin-6 before Biologic Treatment Are Related to Clinical Response in the Patients of Rheumatoid Arthritis

Background Biologics (BIO) have been developed the treatment of rheumatoid arthritis represent a remarkable advance over and beyond the conventional disease-modifying antirheumatic drugs (cDMARDs)1. However, 20–30% of rheumatoid arthritis (RA) patients using biologics are nonresponders or show only minor improvement. Objectives Recently it was reported the concentration of serum cytokine was related the response of treatment for RA2,3. We investigate the relationships of it before taking first BIO and the change of disease activity in our series. Methods One hundred nighty-nine RA patients have received BIO in our institutes from 2004 to 2013. Forty-one patients in BIO naive cases (infliximab 16 cases, etanercept 13, tocilizumab 9, adalimumab 3) were examined the concentration of serum tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6 before receiving first BIO by ELISA. They are estimated the transition of disease activity score (DAS)28/C-reactive protein (CRP) score (4) and clinical disease activity index (CDAI) one year after first BIO. They are 7 men and thirty-four women. Their mean age is 55 years (range 32-74), mean duration of RA affliction was 7.8 years (1-22). Results The mean concentration of serum TNF-alpha and IL-beta, IL-6 was 16.2 (0.6-238) pg/ml,; 10 (10-11); 34.1 (0.5-303) before first BIO, respectively. The mean CRP levels, DAS28-CRP (4) and CDAI were 29 g/dl (range, 1–82), 4.9 (1.0-7.2) and 24.8 (0-67) before first BIO, which improved as 7 (0-34) g/dl, 3.1 (10-64) and 10.3 (0-30) one year after first BIO, respectively. EULAR response criteria were used, and there was a good response in twenty-eight patients (68%), a moderate response in eight (20%) and no response in five (12%), respectively, during the treatment period starting at the commencement of the BIO and one year after. Only the value of serum IL-6 was significantly related to the results of EULAR response and CRP before first BIO (p<0.05, Fig. 1). Four case in good response group was satisfied of criteria of remission using CDAI scoring (<2.8). Conclusions The value of serum IL-6 before receiving BIO seems to have the potential marker for predicting the response of BIO treatment in RA patients. References Klareskog L, et al: Lancet 2004; 363:675-681. Takeuchi T, et al: ARD 2012; 71: 1583-1585. Shimamoto K, et al: J Rheumatol 2013; 40:1074-1081. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3522