Yasuo Niki

Macrophage- and neutrophil-dominant arthritis in human IL-1α transgenic mice

To study the effects of IL-1α in arthritis, we generated human IL-1α (hIL-1α). Transgenic mice expressed hIL-1α mRNA in various organs, had high serum levels of hIL-1α, and developed a severe polyarthritic phenotype at 4 weeks of age. Not only bone marrow cells but also synoviocytes from knee joints produced biologically active hIL-1α. Synovitis started 2 weeks after birth, and 8-week-old mice showed hyperplasia of the synovial lining layer, the formation of hyperplastic synovium (pannus) and, ultimately, destruction of cartilage. Hyperplasia of the synovial lining was due to the accumulation of macrophage-like cells expressing F4/80 molecules. hIL-1α was widely distributed in macrophage- and fibroblast-like cells of the synovial lining cells, as well as synovial fluid monocytes. T and B cells were rare in the synovial fluid, and analysis of marker expression suggests that synoviocytes were directly histolytic and did not act as antigen-presenting cells. In the joints of these mice, we found elevated levels of cells of the monocyte/macrophage and granulocyte lineages and of polymorphonuclear neutrophils (PMNs), most of which expressed Gr-1, indicating that they were mature, tissue-degrading PMNs. Cultured synoviocytes and PMNs from these animals overexpress GM-CSF, suggesting that the hematopoietic changes induced by IL-1 and the consequent PMN activation and joint destruction are mediated by this cytokine.

Membrane-Associated IL-1 Contributes to Chronic Synovitis and Cartilage Destruction in Human IL-1α Transgenic Mice

IL-1 molecules are encoded by two distinct genes, IL-1α and IL-1β. Both isoforms possess essentially identical activities and potencies, whereas IL-1α, in contrast to IL-1β, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1α (hIL-1α) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys64 to Ala271 of the hIL-1α amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1α. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1α activity or concentration of serum hIL-1α. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1α, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.

Metal ions induce bone-resorbing cytokine production through the redox pathway in synoviocytes and bone marrow macrophages.

To evaluate the biological reactions to metal ions potentially released from prosthetic implants, we examined the ability of metal ions to produce bone-resorbing cytokines and the underlying mechanism using synoviocytes and bone marrow (BM) macrophages. The cells were incubated with NiCl(2), CoCl(2), CrCl(3) or Fe(2)(SO(4))(3) at optimal concentrations, which are detectable in joint fluid following total joint arthroplasty. The production of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha were enhanced by all metal ions tested as determined by enzyme-linked immunosorbent assay. From the results of electrophoresis mobility shift assay, all metal ions enhanced the DNA-binding activity of nuclear factor kappaB (NF-kappaB), and p50-p65 heterodimers and p50 homodimers were the major subunits. These effects of the metal ions were considerably blocked by pyrrolidine dithiocarbamate (PDTC) known as a radical scavenger. An electron spin resonance study clearly demonstrated the ability of metal ions to generate activated oxygen species (AOS), especially hydroxyl radicals (*OH), which accounts for PDTC-blockade of metal ion-induced NF-kappaB activation and subsequent cytokine production. Taken together, our data raised the possibility that small amounts of metal ions released from prosthetic implants activate synoviocytes and BM macrophages through the AOS-mediated process (i.e. the redox pathway), and contribute to the initiation of osteolysis at the bone-implant interface.

Repair of full-thickness articular cartilage defects using injectable type II collagen gel embedded with cultured chondrocytes in a rabbit model

BackgroundRecently, tissue-engineered chondrocyte transplantation has been tried to treat full-thickness cartilage defects. We developed an injectable type II collagen gel scaffold by chemically reacting type II collagen with polyethylene glycol crosslinker. This type II collagen was prepared from the nasal septa of cattle. In the present study, chondrocytes embedded in type II collagen gel were injected into rabbit full-thickness cartilage defects without a periosteal graft, and the feasibility for clinical application of the gel was evaluated.MethodsChondrocytes were isolated from 1-kg New Zealand white rabbits. A full-thickness articular cartilage defect (5 mm diameter, 4 mm depth) was created on the patellar groove of the femur of 16 male 3-kg New Zealand white rabbits. A type II collagen solution of mixed chondrocytes at a density of 1 × 107 cells/ml was injected and transplanted into the defect in the right knee. The controls were the defect only in the left knee. At 4, 8, 12, and 24 weeks after operation, four cases from each group were evaluated macroscopically and histologically.ResultsAfter injection into the cartilage defect, the gel bonded to the adjacent cartilage and bone within several minutes. Macroscopic examination revealed that the surface of the transplanted area was smooth and exhibited similar coloration and good integration with the surrounding cartilage at 12 and 24 weeks after transplantation. Histological examination at 8 weeks revealed favorable hyaline cartilage regeneration with good chondrocyte morphology. At 12 and 24 weeks, reparative cartilage remained rich in type II collagen. According to O’Driscoll histological scores, significant differences between the transplanted and control groups were apparent at 12 and 24 weeks. Immunohistochemical staining indicated sufficient type II collagen synthesis in regenerated cartilage 8 weeks after transplantation, and it was maintained until 24 weeks.ConclusionsThese results indicate that type II collagen gel is suitable for injection into cartilage defects without any covering of a graft and offers a useful scaffold during chondrocyte transplantation.

Is minimally invasive surgery in total knee arthroplasty really minimally invasive surgery

Minimization of soft-tissue damage is one of the primary purposes behind the application of minimally invasive surgery (MIS) in total knee arthroplasty (TKA). A consecutive series of 147 TKAs were enrolled in the present study, with 96 MIS-TKAs using 11 quadriceps-sparing, 46 subvastus, 32 midvastus, and 7 parapatellar approaches and 51 conventional TKAs using 22 subvastus, 9 midvastus, and 20 parapatellar approaches. Serum levels of creatinine phosphokinase, myoglobin, aldolase, lactate dehydrogenase, glutamic oxaloacetic transaminase, and creatinine were measured on postoperative days 0, 1, 2, 4, 7, and 14. Postoperative rising index (RI) was expressed as a proportion of the preoperative value. When RIs were compared between MIS-TKA and conventional TKA, no significant differences were found for any enzymes. Interestingly, the midvastus approach displayed the highest RIs for creatinine phosphokinase and myoglobin between the 4 vastus-splitting approaches. Consequently, degree of muscle damage was equivalent between MIS-TKA and conventional TKA, whereas types of vastus-splitting approach appeared closely related to muscle damage.

Rheumatoid arthritis: a risk factor for deep venous thrombosis after total knee arthroplasty? Comparative study with osteoarthritis

BackgroundRecent advances in the understanding of blood coagulation processes favor an inflammatory basis for thrombotic events. In this study, thrombotic risk after total knee arthroplasty (TKA) was assessed and compared between patients with rheumatoid arthritis (RA) and those with osteoarthritis (OA).MethodsSubjects comprised 199 patients (238 knees) with RA and 156 patients (169 knees) with OA. Serum D-dimer levels were measured before and after the operation. Lowdose unfractionated heparin was given for 7 days when patients had a history of previous venous thromboembolism or had a D-dimer level or ≥10 μg/ml of D-dimer on postoperative day 1. Doppler ultrasonography (DUS) was routinely performed preoperatively and on postoperative day (POD) 7 for diagnosing a deep venous thrombosis (DVT).ResultsD-dimer levels on PODs 0, 1, and 7 were, respectively, 4.6, 37.2, and 11.2 μg/ml for RA and 1.8, 42.3, and 13.6 μg/ml for OA. The incidence of DUS-confirmed DVT was 20.6% in the RA group and 43.2% in the OA group, indicating a much higher incidence of postoperative DVT in OA patients (P < 0.001). Interestingly, when patients taking nonsteroidal antiinflammatory drugs (NSAIDs) or those >65 years of age were excluded, the incidence of DVT was comparable in the RA and OA groups. Symptomatic pulmonary embolism and DVT occurred in two and one OA patients and in one and two RA patients, respectively, with one postdischarge DVT included in each group.ConclusionsThe present study revealed that the incidence of DVT following TKA was significantly lower in RA patients than in those with OA. However, when the patients were matched for age and NSAID use, the incidence of DVT was equivalent in the two groups. These findings may allow us to reconsider a prophylactic regimen for venous thromboembolism in patients with RA.

The effect of timing in the administration of hepatocyte growth factor to modulate BMP-2-induced osteoblast differentiation

Development of bone morphogenetic protein (BMP) signaling modulators may provide useful therapeutic options for the treatment of large bony defects in clinical settings. Controversy remains over whether hepatocyte growth factor (HGF) is a positive or negative modulator of BMP-induced osteogenesis. This study analyzed osteogenic properties of HGF, particularly during BMP-2-induced bone formation. Using a mouse model of ectopic bone formation, HGF-impregnated gelatin sponges displayed significantly reduced bone formation induced by BMP-2, both radiologically and histologically. Abrogation of endogenous HGF production by knockdown of HGF mRNA resulted in upregulation of BMP-2-induced ALP activity for C2C12 myoblasts in vitro. In contrast, addition of exogenous HGF inhibited BMP-2-induced ALP activity and osteocalcin production by mouse embryonic fibroblasts (MEFs) through HGF-c-Met interactions. Inhibition of ALP activity by HGF was rescued by U0126, a MEK1/2 inhibitor, indicating that HGF suppresses the BMP-2-Smad axis via activation of ERK1/2. Importantly, treatment with HGF prior to administration of BMP-2 induced cellular proliferation of MEFs and did not influence subsequent osteoblast differentiation induced by BMP-2. The effects of HGF may differ according to the differentiation stage of mesenchymal stem cells, which would explain the inconsistencies seen in osteogenic properties of HGF in previous reports. The timing of HGF treatment is critical and should be carefully determined for successful induction of bone formation by BMPs.

IL-32-PAR2 axis is an innate immunity sensor providing alternative signaling for LPS-TRIF axis

Interleukin (IL)-32 is known to exert adujvant effects on innate immune response, however, receptors and downstream signaling pathways remain to be clarified. Here we found that IL-32γ upregulated serine protease activity of proteinase-3 (PR3), in turn triggering protease-activated receptor 2 (PAR2) signaling. Interestingly, silencing of PR3 or PAR2 using siRNA markedly diminished IL-32γ-induced TNFα and IFN-β mRNA expression. IL-32γ-PAR2 axis utilized TRIF and Ras-Raf-1 pathways. On stimulation with lipopolysaccharide (LPS), differential activation of protein kinase C isoforms modulated the balance between LPS-TLR4-TRIF and IL-32-PAR2-TRIF axes, because LPS was a strong inducer of IL-32γ. IL-32-PAR2-TRIF axis might serve not only as an extracellular sensor of bacterial and autologous proteases, but also as a modulator of innate and adaptive immunity during infection.

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

IntroductionThe present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.MethodsIL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).ResultsSingle injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.ConclusionsThese results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.

Accuracy of Implant Positioning for Minimally Invasive Total Knee Arthroplasty in Patients With Severe Varus Deformity

Minimally invasive surgery (MIS) in total knee arthroplasty (TKA) reportedly yields decreased patient morbidity and a rapid return of function, but how much deformity can be accepted for MIS-TKA remains unclear. This study investigated 238 knees from 218 consecutive patients who underwent MIS-TKA. Patients were divided into groups with tibiofemoral mechanical axis (TFM) 195 degrees or greater and TFM less than 195 degrees, then clinical and radiographic results were compared. Similar improvements in knee score at 3 months postoperatively were obtained in the both groups, whereas radiographic accuracy of the coronal alignment in the TFM >or=195 degrees group was inferior to that in TFM <195 degrees group. Postoperative TFM was significantly worsened in patients with lateral bowing angle of the femoral shaft (LBFS) 4 degrees or greater, and 53% of patients in the TFM >or=195 degrees group displayed LBFS 4 degrees or greater, explaining the inferior radiographic accuracy in this group compared with the TFM <195 degrees group. These results indicate that use of MIS techniques decreases radiographic accuracy, particularly in patients with severe genu varum and increased LBFS.