Inflammatory Osteoclastogenesis Research Articles

Inhibitory effects of luteolin on titanium particle-induced osteolysis in a mouse model

Wear particles liberated from the surfaces of an implanted prosthesis are associated with peri-implant osteolysis and subsequent aseptic loosening. In the latter wear particle-induced inflammation and osteoclastogenesis have been identified as critical factors, and their inhibition as important steps in the treatment of affected patients, such as those undergoing total hip replacement. In this study the ability of luteolin to inhibit both titanium (Ti) particle-induced osteoclastogenesis in vitro and osteolysis in a murine calvaria Ti particle-induced model of osteolysis was examined. The results showed that luteolin, a highly potent and efficient inhibitor of tumor necrosis factor α (TNF-α) and interleukin-6 expression, inhibited Ti particle-induced inflammatory cytokine release, osteoclastogenesis, and bone resorption in bone marrow macrophages. Microcomputed tomography and histological analyses showed that the Ti particles caused significant bone resorption and increased TRAP+ multinuclear osteoclasts in the murine calvarial model of osteolysis, whereas this was not the case in the luteolin treatment group, in which osteolytic suppression was accompanied by a decrease in both TNF-α production and serum levels of the osteoclast marker the C-terminal telopeptide fragment of type I collagen. These results support the use of luteolin as a natural compound in the prevention and treatment of aseptic loosening after total replacement arthroplasty.

TNF-induced osteoclastogenesis and inflammatory bone resorption are inhibited by transcription factor RBP-J.

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of inflammatory bone resorption and associated morbidity in diseases such as rheumatoid arthritis and periodontitis. Mechanisms that regulate the direct osteoclastogenic properties of TNF to limit pathological bone resorption in inflammatory settings are mostly unknown. Here, we show that the transcription factor recombinant recognition sequence binding protein at the J(κ) site (RBP-J) strongly suppresses TNF-induced osteoclastogenesis and inflammatory bone resorption, but has minimal effects on physiological bone remodeling. Myeloid-specific deletion of RBP-J converted TNF into a potent osteoclastogenic factor that could function independently of receptor activator of NF-κB (RANK) signaling. In the absence of RBP-J, TNF effectively induced osteoclastogenesis and bone resorption in RANK-deficient mice. Activation of RBP-J selectively in osteoclast precursors suppressed inflammatory osteoclastogenesis and arthritic bone resorption. Mechanistically, RBP-J suppressed induction of the master regulator of osteoclastogenesis (nuclear factor of activated T cells, cytoplasmic 1) by attenuating c-Fos activation and suppressing induction of B lymphocyte-induced maturation protein-1, thereby preventing the down-regulation of transcriptional repressors such as IRF-8 that block osteoclast differentiation. Thus, RBP-J regulates the balance between activating and repressive signals that regulate osteoclastogenesis. These findings identify RBP-J as a key upstream negative regulator of osteoclastogenesis that restrains excessive bone resorption in inflammatory settings.

Lysine392, a K63‐linked ubiquitination site in NEMO, mediates inflammatory osteoclastogenesis and osteolysis

PMMA particles released from bone implants are considered major contributor to osteolysis and subsequent implant failure. Although the ensuing inflammatory response has been described, the mechanisms underlying PMMA particulate-induced osteolysis remain enigmatic. In previous studies, we have established that activation of Nuclear factor kappa-B (NF-κB) and MAP kinase pathways plays a central role in the pathogenesis of inflammatory osteolysis. Specifically, we have shown that impeding IKK complex assembly, and thus subsequent NF-κB activation, dampens particle-induced osteolysis. The IKK complex consists of IKKα, IKKβ, and IKKγ, also known as NEMO. NEMO has no catalytic activity and serves as a scaffold protein facilitating assembly and distal activation of NF-κB signaling. In fact, blocking binding of NEMO with IKKα/β abolishes NF-κB activity. In the current study, we identify Lysine 392 residue in NEMO as crucial mediator of PMMA particle-induced inflammatory osteoclastogenesis and osteolysis. Using mice in which NEMO-K392R mutation has been introduced, we provide evidence that PMMA-induced osteoclasts and osteolytic responses are impaired. Furthermore, we show that this impairment is likely due to poor activation of NF-κB and Erk, but not other MAP kinases. Our findings suggest that NEMO Lysine392, a well-established K63-linked polyubiquitination site, is an important mediator of PMMA-induced osteolysis. Therefore, this NEMO motif should be considered as a target to combat PMMA particle-induced osteolysis.

T10c12‐CLA maintains higher bone mineral density during aging by modulating osteoclastogenesis and bone marrow adiposity

Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier, we showed that CLA (equal mixture of c9t11-CLA and t10c12-CLA) could protect age-associated bone loss by modulating inflammatory markers and osteoclastogenesis. Since, c9t11-CLA and t10c12-CLA isomers differentially regulate functional parameters and gene expression in different cell types, we examined the efficacy of individual CLA isomers against age-associated bone loss using 12 months old C57BL/6 female mice fed for 6 months with 10% corn oil (CO), 9.5% CO + 0.5% c9t11-CLA, 9.5% CO + 0.5% t10c12-CLA or 9.5% CO + 0.25% c9t11-CLA + 0.25% t10c12-CLA. Mice fed a t10c12-CLA diet maintained a significantly higher bone mineral density (BMD) in femoral, tibial and lumbar regions than those fed CO and c9t11-CLA diets as measured by dual-energy-X-ray absorptiometry (DXA). The increased BMD was accompanied by a decreased production of osteoclastogenic factors, that is, RANKL, TRAP5b, TNF-alpha and IL-6 in serum. Moreover, a significant reduction of high fat diet-induced bone marrow adiposity was observed in t10c12-CLA fed mice as compared to that of CO and c9t11-CLA fed mice, as measured by Oil-Red-O staining of bone marrow sections. In addition, a significant reduction of osteoclast differentiation and bone resorbing pit formation was observed in t10c12-CLA treated RAW 264.7 cell culture stimulated with RANKL as compared to that of c9t11-CLA and linoleic acid treated cultures. In conclusion, these findings suggest that t10c12-CLA is the most potent CLA isomer and it exerts its anti-osteoporotic effect by modulating osteoclastogenesis and bone marrow adiposity.

Protective effects of COX-2 inhibitor on titanium-particle-induced inflammatory osteolysis via the down-regulation of RANK/RANKL

Particle-wear-induced inflammatory osteolysis remains a major problem for the long-term success of total joint arthroplasty. Previous studies have demonstrated that cyclooxygenase-2 (COX-2) is expressed abundantly in the tissue around a failed implant. However, the role of COX-2 in the development of particle-wear-induced osteoclastogenesis remains unclear. The aim of the study was to test the hypothesis that Dynastat, a COX-2 inhibitor, ameliorates particle-wear-induced inflammatory osteoclastogenesis through the down-regulation of the receptor activators of nuclear factor-κB (RANK) and nuclear factor-κB ligand (RANKL) expression in a murine osteolysis model. Titanium (Ti) particles were introduced into established air pouches in BALB/c mice, followed by the implantation of calvaria bone from syngeneic littermates. Dynastat was given to mice intraperitoneally 2days before the introduction of Ti particles and maintained until the mice were sacrificed. Pouch tissues were collected 14days after Ti inoculation for molecular and histological analysis. The results showed that Dynastat has more impact on Ti-particle-induced prostaglandin E2 expression and less on the expression of interleukin-1β and tumor necrosis factor-α. Dynastat inhibited Ti-particle-induced osteoclastogenesis by reducing the gene activation of RANK and RANKL, and diminishing the RANKL expression in Ti-particle-charged pouches. Dynastat markedly reduced the number of tartrate-resistant acid-phosphatase-positive cells in pouch tissues stimulated by Ti particles. In conclusion, this study provides evidence that Dynastat can markedly inhibit Ti-particle-induced osteoclastogenesis by the down-regulation of RANK/RANKL in a murine air pouch model, and is a promising therapeutic candidate for the treatment of inflammatory osteolysis induced by wear particles.

Dual role of interleukin-17 in pannus growth and osteoclastogenesis in rheumatoid arthritis

IntroductionIn a murine model, interleukin (IL)-17 plays a critical role in the pathogenesis of arthritis. There are controversies, however, regarding whether IL-17 is a proinflammatory mediator in rheumatoid arthritis (RA). We previously established an ex vivo cellular model using synovial tissue (ST)-derived inflammatory cells, which reproduced pannus-like tissue growth and osteoclastic activity in vitro. Using this model, we investigated the effects of IL-17 on pannus growth and osteoclastogenesis in RA.MethodsInflammatory cells that infiltrated synovial tissue from patients with RA were collected without enzyme digestion and designated as ST-derived inflammatory cells. ST-derived inflammatory cells were cultured in the presence or absence of IL-17 or indomethacin, and the morphologic changes were observed for 4 weeks. Cytokines produced in the culture supernatants were measured by using enzyme-linked immunosorbent assay kits. Osteoclastic activity was assessed by the development of resorption pits in calcium phosphate-coated slides.ResultsExogenous addition of IL-17 dramatically enhanced the spontaneous production of IL-6 and prostaglandin E2 (PGE2) by the ST-derived inflammatory cells, while it had no effect on the production of tumor necrosis factor (TNF)-α and macrophage colony-stimulating factor (M-CSF). Furthermore, IL-17 did not affect the spontaneous development of pannus-like tissue growth and osteoclastic activity by the ST-derived inflammatory cells. On the other hand, IL-17 enhanced pannus-like tissue growth, the production of TNF-α and M-CSF and the development of osteoclastic activity in the presence of indomethacin, an inhibitor of endogenous prostanoid production, while exogenous addition of PGE1 suppressed their activities.ConclusionsThe present study suggests that IL-17 induces negative feedback regulation through the induction of PGE2, while it stimulates proinflammatory pathways such as inflammatory cytokine production, pannus growth and osteoclastogenesis in RA.

Polymethylmethacrylate and titanium alloy particles activate peripheral monocytes during periprosthetic inflammation and osteolysis

We investigated the interactions of particulate PMMA or titanium alloy, patient blood monocytes, and periprosthetic tissues using a SCID-hu model of aseptic loosening. Periprosthetic tissues and bone chips obtained at revision surgery for loosening were transplanted into muscles of SCID mice. Peripheral blood monocytes (PBMCs) isolated from the same donors were fluorescently labeled and co-cultured with PMMA or Ti-6Al-4V particles before intraperitoneal injection. Control mice with periprosthetic tissue or non-inflammatory ligament xenografts received naive PBMCs transfusion. Mice were euthanized 2 weeks after PBMC transfusion. The human tissues were well accepted in SCID mice. Transfused fluorescent-labeled PBMCs were markedly accumulated in transplanted periprosthetic tissues. Multinucleated osteoclast-like cells were commonly seen within retrieved xenograft tissue, and focal bone erosions were ubiquitous. Total cell densities and CD68+ cells within the xenograft were significantly increased in mice transfused with PMMA and Ti-provoked PBMCs compared to the naïve PBMC animals (p < 0.05). Immunohistochemical staining identified much stronger positive IL-1 and TNF stains in xenografts from either PMMA or Ti-stimulated monocytes transfusion groups (p < 0.05). TRAP+ cells were found around bone chips in both activated-PBMCs groups, although markedly more aggregated TRAP+ cells in the PMMA-challenged group than in the titanium group (p < 0.05). MicroCT assessment confirmed the significant decrease of bone mineral density in chips interacted with activated-monocytes/osteoclasts. In conclusion, PMMA or titanium particles readily activate peripheral monocytes and promote the cell trafficking to the debris-containing prosthetic tissues. Particles-provoked PBMCs participated in and promoted the local inflammatory process, osteoclastogenesis, and bone resorption.

Adrenomedullin inhibits MAPK pathway-dependent rheumatoid synovial fibroblast-mediated osteoclastogenesis by IL-1 and TNF-α

The objective of this study is to determine the effects of adrenomedullin (AM) on IL-1- and TNF-alpha-induced rheumatoid synovial fibroblasts (RASFs)-mediated osteoclastogenesis. The formation of osteoclasts in co-cultures of RASFs and peripheral blood mononuclear cells was evaluated by tartrate-resistant acid phosphatase and resorption pit formation assay. The expression of RANKL, OPG, p-ERK, p-p38, and p-JNK was examined by immunoblotting and quantitative reverse transcription-polymerase chain reaction. AM (1-52) inhibits IL-1- and TNF-alpha-induced RASFs-mediated osteoclastogenesis. AM affected IL-1-, TNF-alpha-induced RANKL and OPG expression in RASFs. AM also inhibits IL-1 and TNF-alpha-induced phosphorylation of ERK-1/2, p38 MAPK, and JNK. Inhibitor of AM (AM 22-52) inhibits the effects of AM on the osteoclastogenesis. These results suggest that AM might be involved in the inflammatory cytokines-mediated osteoclastogenesis and thus bone damage, and indicate that it can be a new therapeutic strategy against joint destruction in RA.

T10c12 CLA isomer prevents age associated bone loss by modulating osteoclastogenesis

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age‐related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier we have shown that CLA (equal ratio of c9t11 and t10c12 isomers) can protect age associated bone loss by modulating inflammatory markers and osteoclastogenesis. However, c9t11 and t10c12 CLA individual isomers differentially regulate functional parameters and gene expression in different cell types. Therefore, the present study was undertaken to examine the differential effect of c9t11 and t10c12 CLA isomers on bone mineral density (BMD) in middle‐aged (12 mo) C57BL/6 female mice. After 5 months on diet, t10c12 CLA‐isomer fed mice maintained a significantly higher BMD in femoral, tibial and lumbar regions than c9t11 and corn oil (CO)‐fed mice. The increased BMD was accompanied by a decreased activity of osteoclastogenic factor, RANKL and decreased osteoclast activity as measured by TRAP5b in serum. A significant reduction of osteoclasts differentiation was also observed in RANKL stimulated RAW 264.7 cells in t10c12 CLA isomer treated culture as compared to c9t11 CLA isomer or linoleic acid treated culture. In conclusion, these findings suggest that t10c12 isomer is the most potent CLA isomer to prevent the bone loss by modulating osteoclastogenesis. Research support: NIH R21 AG027562.

Inflammatory osteoclastogenesis can be induced by GM-CSF and activated under TNF immunity

In inflammatory arthritis such as RA, osteoclastic activity is severely enhanced. GM-CSF was reportedly elevated in synovial fluid, but is a strong inhibitor of osteoclastogenesis; here lies a contradiction. Our objective was to examine what type of osteoclasts generate and resorb bone with resistance to GM-CSF in an inflammatory joint. Monocyte-derived cells generated in GM-CSF were morphologically and immunophenotypically different from both the conventional DC and macrophage. They could differentiate into osteoclasts in the presence of RANKL + M-CSF, acquiring a stronger osteoclastic activity under TNF treatment. Furthermore, their differentiation was not inhibited by GM-CSF, while monocyte-derived osteoclast differentiation was completely inhibited. The resorption was suppressed by GM-CSF, and the existence of another osteoclastic pathway has been suggested. Our findings indicate another type of osteoclast exists in inflammatory arthritis.

A p38 Mitogen‐Activated Protein Kinase Inhibitor Arrests Active Alveolar Bone Loss in a Rat Periodontitis Model

Gram-negative bacterial species, such as Actinobacillus actinomycetemcomitans, contain lipopolysaccharide (LPS) that initiates the innate immune system, resulting in inflammatory alveolar bone loss. LPS activates Toll-like receptors on membrane surfaces, stimulating many intracellular signaling cascades, including the p38 mitogen-activated protein kinase (MAPK). Activation of p38 signaling mediates inflammatory cytokine expression, contributing toward osteoclastogenesis and bone loss. The aim of this study was to determine whether the novel, orally active p38 MAPK inhibitor SD282 could arrest progression of LPS-induced alveolar bone destruction in rats. Three groups of female Sprague-Dawley rats received LPS injections to the palatal molar gingiva three times per week for 4 weeks to establish periodontitis. From weeks 5 through 8, two groups received the drug SD282 (N = 14) or 1% polyethylene glycol drug vehicle (N = 14) via oral gavage in addition to LPS injections. The third group continued to receive only LPS injections (N = 8). Microcomputed tomography was used to measure volumetric alveolar bone loss, expressed as bone volume fraction (BVF). Expression of interleukin (IL)-1 and -6 and tumor necrosis factor-alpha (TNF-alpha) was assessed by immunohistochemistry, and osteoclasts were enumerated by tartrate-resistant acid phosphatase staining. By 4 weeks, severe alveolar bone resorption was seen in LPS-injected animals. Administration of SD282 significantly blocked additional volumetric bone loss in the LPS-only versus LPS + SD282 groups (0.37 +/- 0.01 BVF versus 0.43 +/- 0.01 BVF; P < 0.01). Significant reductions in IL-1beta (P < 0.01 ), TNF-alpha (P < 0.05), and osteoclast formation (P < 0.01) occurred in the presence of SD282. An orally active p38 MAPK inhibitor reduced LPS-induced inflammatory cytokine expression, osteoclastogenesis, and alveolar bone loss in rats. Within the limits of the current study, SD282 arrested periodontal disease progression, thus highlighting the therapeutic potential of this novel class of inhibitors.

Inhibition of SFRP1 Reduces Severity of Periodontitis

Porphyromonas gingivalis (P. gingivalis) is implicated as a major pathogen in periodontitis, a common infectious disease characterized by the inflammation and destruction of periodontal tissues. Secreted frizzled-related protein 1 (SFRP1) modulates apoptosis in different cell types. To characterize the roles of SFRP1 in periodontitis, we used a P. gingivalis-induced murine periodontitis model. Inflammatory responses were measured by morphometric and histomorphometric analysis, apoptosis assay, and immunohistochemistry. We found that P. gingivalis-infected mouse periodontal tissues expressed significantly more SFRP1 compared with those of control mice. Also, in P. gingivalis-infected animals, more apoptosis of inflammatory cells, fibroblasts, and bone-lining cells was observed compared with controls. Antibody experiments aimed at inhibiting SFRP1 expression in periodontitis resulted in a reduction of periodontal breakdown, inflammatory cell infiltrate, osteoclastogenesis, and apoptosis of inflammatory cells and fibroblasts. The results of our studies suggest that SFRP1 may be involved in the development of periodontitis, since inhibiting SFRP1 resulted in reduced periodontal breakdown.

Osteoclasts: What Do They Do and How Do They Do It?

As Americans live longer, degenerative skeletal diseases, such as osteoporosis, become increasingly prevalent. Regardless of cause, osteoporosis reflects a relative enhancement of osteoclast activity. Thus, this unique bone resorptive cell is a prominent therapeutic target. A number of key observations provide insights into the mechanisms by which precursors commit to the osteoclast phenotype and how the mature cell degrades bone. The osteoclast is a member of the monocyte/macrophage family that differentiates under the aegis of two critical cytokines, namely RANK ligand and M-CSF. Tumor necrosis factor (TNF)-alpha also promotes osteoclastogenesis, particularly in states of inflammatory osteolysis such as that attending rheumatoid arthritis. Once differentiated, the osteoclast forms an intimate relationship with the bone surface via the alphavbeta3 integrin, which transmits matrix-derived, cytoskeleton-organizing, signals. These integrin-transmitted signals include activation of the associated proteins, c-src, syk, Vav3, and Rho GTPases. The organized cytoskeleton generates an isolated microenvironment between the cell's plasma membrane and the bone surface in which matrix mineral is mobilized by the acidic milieu and organic matrix is degraded by the lysosomal protease, cathepsin K. This review focuses on these and other molecules that mediate osteoclast differentiation or function and thus serve as candidate anti-osteoporosis therapeutic targets.

Biological variation and reference intervals for circulating osteopontin, osteoprotegerin, total soluble receptor activator of nuclear factor kappa B ligand and high‐sensitivity C‐reactive protein

Objective. Monitoring inflammatory diseases and osteoclastogenesis with osteopontin (OPN), osteoprotegerin (OPG), total soluble receptor activator of nuclear factor kappa B ligand (total sRANKL) and high‐sensitivity C‐reactive protein (hsCRP) has recently attracted increased interest. The purpose of our study was to determine reference intervals, variability caused by sampling time, biological variation and stability after repeated freeze–thaw cycles of circulating levels of OPN, OPG, total sRANKL and hsCRP. Material and methods. Plasma OPN and plasma OPG concentrations were determined with sandwich ELISA; serum total sRANKL concentration was determined using a two‐site sandwich ELISA; and hsCRP was analysed by turbidimetry in 300 Danish blood donors (183 M and 117 F) with a median age of 43 years (range 18–64 years). Variability due to biological variation and sampling time was studied in serial samples from 38 healthy subjects. Results. The 95th percentiles in the donors were 76 µg/L for OPN, 4.2 pmol/L for OPG, 40.2 nmol/L for total sRANKL and 12 mg/L for hsCRP. The overall medians for both genders were 51 µg/L, 2.2 pmol/L, 0.66 nmol/L and 1.0 mg/L, respectively. We found a significant correlation between hsCRP and OPN (rho = 0.173; p<0.003). The biological within‐subject variations were calculated to be 8.2 % for OPN, 8.8 % for total sRANKL and 50 % for hsCRP. Conclusions. Reference intervals have been established with a high analytic performance for OPN and an acceptable analytic performance for OPG and total sRANKL. The study revealed low biological variation for OPN and total sRANKL and high biological variation for hsCRP.

Association between UHMWPE particle-induced inflammatory osteoclastogenesis and expression of RANKL, VEGF, and Flt-1 in vivo

Wear debris-induced vascularized granulomatous periprosthetic tissue may augment the progress of prosthetic loosening, a major clinical problem after total joint replacement. The purpose of this study is to investigate the association of ultra-high-molecular-weight polyethylene (UHMWPE) particle-induced inflammatory osteoclastogenesis and expression of RANK/RANKL and VEGF/VEGF receptors (Flt-1 and Flk-1) using a mouse osteolysis model. UHMWPE particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. Mice were injected with either recombinant VEGF or VEGF inhibitor (VEGF R2/F(c) Chimera). Mice without drug treatment, as well as mice injected with saline alone were included. Each group contains 10 mice. Pouch tissues were harvested 2 weeks after bone implantation for histological and molecular analysis. UHMWPE stimulation significantly increased VEGF gene expression, and exerted a lower enhancement effect on the gene expression of Flt-1 and Flk-1. UHMWPE-stimulated VEGF production was markedly reduced by VEGF inhibitor treatment. Immunofluorescent staining indicated that pouch tissue macrophages were the main source of both VEGF and Flt-1 production. A positive association was observed between tissue inflammation and the levels of VEGF and Flt-1 gene transcripts. Both RANK and RANKL gene transcripts were significantly increased by UHMWPE stimulation, which was subsequently reduced by VEGF inhibitor treatment (p<0.05). VEGF treatment increased TRAP(+) cells in pouches either with or without UHMWPE particle stimulation, and VEGF inhibitor treatment caused a significant reduction in the number of TRAP(+) cells in UHMWPE-containing pouches. This study suggests that VEGF has a role in the regulation of RANK/RANKL-mediated osteoclastogenesis, and warrant future investigations to elucidate the role of VEGF signaling in the pathogenesis of prosthetic loosening.

B Cells Play an Important Role in Lipopolysaccharide-Induced Bone Resorption

The host immune system, especially activated T cells, plays a crucial role in inflammatory bone resorption and osteoclastogenesis. Previously, we showed that T cells are involved in inflammatory bone resorption in vivo. However, little is known about whether B cells are involved in inflammatory bone resorption and how B cells take part in osteoclastogenesis. Therefore, the aim of this study was to examine whether B c ells truly influence inflammatory bone resorption in vivo. Alveolar bone resorption in normal mice, in SCID mice that lack both B and T cells, and in B cell-reconstituted SCID mice was compared histopathologically after repeated injections of lipopolysaccharide (LPS) into the gingiva. Furthermore, we examined whether the B cells that are stimulated by LPS are involved in osteoclastogenesis in vitro. As a result, the B cell-reconstituted SCID mice showed stronger inflammatory bone resorption than the SCID mice. Also, in vitro, LPS-stimulated B cells enhanced osteoclastogenesis and anti-tumor necrosis factor (TNF)-alpha antibody completely blocked osteoclastogenesis induced by LPS-stimulated B cells. These results suggest that B cells promote inflammatory bone resorption through TNF-alpha.

Interleukin-6: a new therapeutic target

The therapeutic success of biological agents, especially the tumour necrosis factor (TNF) inhibitors, has opened a new chapter in the book of therapies for rheumatoid arthritis. Nevertheless, more than 50% of patients may not respond by > 50% improvement. New compounds have recently entered the treatment arena. One of these is rituximab, which depletes B cells, and another, abatacept, interferes with T-cell co-stimulation. However, although these agents may be effective in a number of patients who fail to respond to TNF blockade, they only rarely induce remission and overall 50% response rates do not exceed those with the TNF inhibitors. Among the major proinflammatory cytokines, IL-6 plays a pleiotropic role both in terms of activating the inflammatory response and osteoclastogenesis. Here, we review recent phase II trials of tocilizumab, a humanized anti-IL-6 receptor antibody that achieves a significant therapeutic response rate.

Erythromycin inhibits wear debris‐induced inflammatory osteolysis in a murine model

Up to 20% of patients with total joint arthroplasty will develop radiographic evidence of aseptic loosening (AL), which most likely results from an inflammatory response to billions of wear debris shed from the implant. Our previous work has demonstrated that erythromycin (EM), a macrolide antibiotic, inhibits wear debris-induced inflammatory osteoclastogenesis through the reduction of cytokine production and osteoclast differentiation, both of which involve the NF-kappaB pathway. The aim of the current study was to determine whether EM inhibits wear debris-induced inflammatory osteolysis in a murine osteolysis model. Ultrahigh molecular-weight polyethylene (UHMWPE) debris was introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. EM (2 mg/kg/day) was given to mice intraperitoneally 2 days before UHMWPE introduction and maintained until the sacrifice of the mice. Mice with and without EM treatment, as well as control mice injected with saline alone were included in this study. Pouch tissues were collected 14 days after UHMWPE inoculation for molecular and histology analysis. Our findings indicate that: (1) EM reduced UHMWPE-induced tissue inflammation, including the diminished pouch membrane thickness, reduced inflammatory cellular infiltration, and lowered IL-1beta and TNF-alpha expression (mRNA and protein); (2) EM inhibited UHMWPE-induced osteoclastogenesis, with reduced gene activation of RANK, RANKL, and CPK, and diminished RANKL expression in UHMWPE stimulated pouches, and (3) EM markedly reduced the number of TRAP(+) cells in pouch tissues, and protected against bone collagen depletion. In conclusion, this study provides the evidence that EM inhibits the UHMWPE particles-induced inflammatory osteolysis in a murine model, and represents a promising therapeutic candidate for the prevention and treatment of AL.

TNF receptor type 1 regulates RANK ligand expression by stromal cells and modulates osteoclastogenesis.

TNFalpha is a major osteoclastogenic cytokine and a primary mediator of inflammatory osteoclastogenesis. We have previously shown that this cytokine directly targets osteoclasts and their precursors and that deletion of its type-1 receptor (TNFr1) lessens osteoclastogenesis and impacts RANK signaling molecules. Osteoclastogenesis is primarily a RANK/RANKL-dependent event and occurs in an environment governed by both hematopoietic and mesenchymal compartments. Thus, we reasoned that TNF/TNFr1 may regulate RANKL and possibly RANK expression by stromal cells and osteoclast precursors (OCPs), respectively. RT-PCR experiments reveal that levels of RANKL mRNA in WT stromal cells are increased following treatment with 1,25-VD3 compared to low levels in TNFr1-null cells. Expression levels of OPG, the RANKL decoy protein, were largely unchanged, thus supporting a RANKL/OPG positive ratio favoring WT cells. RANK protein expression by OCPs was lower in TNFr1-null cells despite only subtle differences in mRNA expression in both cell types. Mix and match experiments of different cell populations from the two mice phenotypes show that WT stromal cells significantly, but not entirely, restore osteoclastogenesis by TNFr1-null OCPs. Similar results were obtained when the latter cells were cultured in the presence of exogenous RANKL. Altogether, these findings indicate that in the absence of TNFr1 both cell compartments are impaired. This was further confirmed by gain of function experiments using TNFr1- null cultures of both cell types at which exogenous TNFr1 cDNA was virally expressed. Thus, restoration of TNFr1 expression in OCPs and stromal cells was sufficient to reinstate osteoclastogenesis and provides direct evidence that TNFr1 integrity is required for optimal RANK-mediated osteoclastogenesis.

Erythromycin inhibits wear debris-induced osteoclastogenesis by modulation of murine macrophage NF-κB activity

Activation of nuclear factor kappa B (NF-κB) signaling in response to cell stimulation by wear debris may be critical in the pathogenesis of aseptic loosening. Erythromycin (EM), a macrolide antibiotic, has been shown to effectively suppress some types of inflammatory reactions. In this study, we examined the effect of EM on wear debris-induced osteoclastic bone resorption in vitro. EM inhibited Ca + release from neonatal calvaria co-cultured with conditioned medium from mouse RAW264.7 macrophages activated by wear debris. Inhibition of Ca + release was associated with a decreased number of tartrate-resistant acid phosphatase (TRAP)-positive cells in cultured bones. To investigate the mechanism whereby EM inhibits bone-resorption, RAW cells were incubated with wear debris in the presence EM. Real time RT-CR analysis revealed that EM (5 μg/ml) significantly inhibited mRNA expression of NF-κB, cathepsin K (CPK), IL-1β and TNFα, but not RANK in RAW cells stimulated with wear debris. Furthermore, electrophoretic mobility-shift assay showed that EM (0.2 μg–5 μg/ml) could reduce DNA-binding activity of NF-κB in RAW cells stimulated with wear debris. The inhibition of inflammatory osteoclastogenesis by EM treatment was further confirmed by an osteoclast (OC) formation assay using primary cultures of mouse bone marrow progenitor cells stimulated with macrophage colony-stimulating factor and RANK ligand (RANKL). EM treatment (5 μg/ml) resulted in more than 70% reduction in multinucleated OC formation and 50% reduction of TRAP + cells by bone marrow progenitor cells. Our findings support that EM suppresses wear debris-induced osteoclastic bone resorption by, at least, down-regulation of NF-κB signaling pathway. It appears that EM represents a potential therapeutic candidate for the treatment and prevention of aseptic loosening.