Suppressors of cytokine signaling-1 and -3 regulate osteoclastogenesis in the presence of inflammatory cytokines

Bone metabolism and the immune system have a correlative relationship, and both are controlled by various common cytokines, such as IFNs and ILs, produced in the bone microenvironments. The suppressor of cytokine signaling-1 (SOCS1) and SOCS3 are negative regulators of such cytokines. Although SOCSs are shown to be induced during osteoclast differentiation, their physiological roles in osteoclast differentiation and function have not been clarified. Thus, we examined the roles of SOCS1 and SOCS3 in osteoclastogenesis using SOCS1- and SOCS3-deficient mice. IFN-gamma-mediated inhibition of osteoclast differentiation from bone marrow-derived monocytes (BMMs) was strongly enhanced in SOCS1-deficient BMMs, but was diminished in SOCS1-overexpressing BMMs. Moreover, LPS-induced osteoclastogenesis and bone destruction in vivo were suppressed in SOCS1(+/-) mice compared with those in wild-type mice, suggesting that SOCS1 antagonizes the inhibitory effect of IFN-gamma on osteoclastogenesis. SOCS3 did not alter the inhibitory effect of IFNs in osteoclastogenesis in both gain and loss of functional assays; however, the suppressive effect of IL-6 on osteoclast differentiation was greater in SOCS3-deficient BMMs than in wild-type BMMs in vitro. In addition, IL-6 significantly prevented LPS-induced bone destruction in SOCS3-deficient mice, although it failed in wild-type mice in vivo. In SOCS3-deficient BMMs, expression levels of TNF-receptor-associated factor-6 and IkappaB were drastically reduced and receptor activator of the NF-kappaB ligand-induced IkappaB phosphorylation was severely impaired in the presence of IL-6. These data suggest that both SOCS1 and SOCS3 regulate osteoclastogenesis by blocking the inhibitory effect of inflammatory cytokines on receptor activator of the NF-kappaB ligand-mediated osteoclast differentiation signals. Selective suppression of SOCS1 and SOCS3 in osteoclast precursors may be a possible therapeutic strategy for inflammatory bone destruction.     

Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

趋化因子CXCL10在心肌细胞及巨噬细胞中的表达机制

目的探讨心肌缺血-再灌注损伤中趋化因子CXCL10的产生机制。方法分别用LPS、H2O2、Ca2+载体A23187刺激原代培养的心肌细胞、骨髓来源的巨噬细胞或二者混合培养的共培养系统后,ELISA检测培养基上清中的趋化因子CXCL10和促炎性细胞因子IL-1β、IL-6、TNF-α的含量,观察其表达动力学。结果①大剂量(10μg/mL)的LPS刺激心肌细胞主要产生趋化因子CXCL10;刺激骨髓来源巨噬细胞主要产生促炎性细胞因子IL-1β、IL-6、TNF-α。②H2 O2、Ca2+通道激活剂并不能使产生趋化因子CXCL10或IL-1β、IL-6、TNF-α这些促炎性细胞因子。③骨髓来源的巨噬细胞促进心肌细胞表达趋化因子CXCL10;心肌细胞促进骨髓来源的巨噬细胞表达IL-6、TNF-α,但抑制IL-1β的表达。结论心肌细胞是心肌缺血-再灌注损伤中CXCL10潜在的细胞来源;CXCL10的表达,主要依赖于TLR4的激活。     

Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor –αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.     

YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m⁶A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m⁶A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/β, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1β and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.     

Identification and characterization of the precursors committed to osteoclasts induced by TNF-related activation-induced cytokine/receptor activator of NF-kappa B ligand

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-kappaB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-gamma, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-kappaB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-alpha in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.     

The phosphatidylinositol 3-kinase-mediated production of interferon-beta is critical for the lipopolysaccharide inhibition of osteoclastogenesis

AIMS: This study was performed to define molecular mechanisms underlying lipopolysaccharide (LPS)-induced inhibition of osteoclastogenesis. MAIN METHODS: The LPS-dependent signaling pathways leading to the inhibition of osteoclastogenesis were examined using chemical inhibitors and neutralizing antibodies. Key findings: Lipopolysaccharide (LPS) has been shown to induce massive bone loss in vivo by stimulating osteoclast differentiation and activity. However, the direct effect of LPS on osteoclastogenesis in vitro is complex. Based on the context of the differentiation state of precursors, LPS has been shown to either augment or inhibit osteoclast differentiation. When added to receptor activator of NF-kappaB ligand (RANKL)-primed precursors, LPS enhances osteoclast differentiation via the production of TNF-alpha. On the other hand, LPS inhibits osteoclastogenesis from early precursors like bone marrow macrophages by hitherto unknown mechanism. In the present study, we investigated the mechanism by which LPS inhibits osteoclastogenesis. We have identified that the phosphatidylinositol 3-kinase (PI 3-kinase) dependent production of IFN-beta and resultant inhibition of c-Fos expression upon LPS stimulation of bone marrow macrophages are responsible for the LPS-induced inhibition of osteoclastogenesis. Inhibition of PI 3-kinase, neutralization of IFN-beta, and overexpression of c-Fos respectively prevented the LPS-induced inhibition of osteoclast differentiation. SIGNIFICANCE: Our results provide a molecular understanding of the differentiation stage-specific dual effect of LPS on osteoclastogenesis.     

Effects of CXCL4/CXCR3 on the lipopolysaccharide-induced injury in human umbilical vein endothelial cells

Chemokines and inflammatory response of endothelial cells is crucial in the development and progression of inflammatory disease. Lipopolysaccharide (LPS) is a well-known factor to trigger inflammatory response and induce damage of endothelial cells. The present study used lipopolysaccharide (LPS)-treated human vascular endothelial cells (HUVECs) to investigate the function of chemokine CXC chemokine ligand 4 (CXCL4) and its receptor CXC chemokine receptor 3 (CXCR3) in inflammatory-induced endothelial injury. LPS exposure (50, 100, 200 ng/ml) to HUVECs induced a dose- and time-dependent increase in CXCL4 and CXCR3 expression at both mRNA and protein levels. The LPS-induced endothelium hyperpermeability was inhibited by the addition of CXCL4 neutralizing antibody. Moreover, the addition of CXCL4 neutralizing antibody abolished the effects of LPS on tight junction (TJ) protein expression (occludin claudin-4 and Zonula occluden-1[ZO-1]) and p38 phosphorylation, which is supported by the observation of increased TJ protein expression and decreased p38 phosphorylation in LPS-treated HUVECs. SB203580, a p38 inhibitor, protected HUVECs from CXCL4-stimulated damage. In conclusion, CXCL4/CXCR3, which was enhanced by LPS, may be involved in endothelial proliferation, apoptosis, and permeability via the p38 signaling pathway.     

Upregulation of MIP-2 (CXCL2) expression by 15-deoxy-Delta(12,14)-prostaglandin J(2) in mouse peritoneal macrophages

A peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), has been reported to possess anti-inflammatory activity in activated monocytes/macrophages. In this study, we investigated the effect of 15d-PGJ(2) on the lipopolysaccharide (LPS)-induced expression of chemokine mRNAs, especially macrophage inhibitory protein (MIP)-2 (CXCL2), in mouse peritoneal macrophages. The inhibitory actions of the natural PPARgamma ligands, 15d-PGJ(2) and prostaglandin A1 (PGA1), on the expression of RANTES (regulated upon activation, normal T expressed and secreted; CCL5), MIP-1beta (CCL4), MIP-1alpha (CCL3), IFN-gamma-inducible protein 10 kilodaltons (IP-10; CXCL10) and monocyte chemoattractant protein-1 (MCP-1; CCL2) mRNA in LPS-treated cells were stronger than those of the synthetic PPARgamma ligands troglitazone and ciglitazone. However, 15d-PGJ(2) enhanced the expression of LPS-induced MIP-2 (CXCL2) mRNA. A specific PPARgamma antagonist (GW9662) had no effect on the inhibitory action of 15d-PGJ(2) and PGA1 in LPS-induced chemokine mRNA expression and on the synergistic action of 15d-PGJ(2) in LPS-induced MIP-2 (CXCL2) expression. Moreover, LPS itself reduced the expression of PPARgamma. Although the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was remarkable, the production of MIP-2 (CXCL2) in cells treated with 15d-PGJ(2) and LPS did not increase compared to the production in cells treated with LPS alone. The synergistic action of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was dependent on the activation of nuclear factor-kappaB (NF-kappaB), and 15d-PGJ(2) increased the phosphorylation of p38 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in cells stimulated with LPS. These results suggest that the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) expression is PPARgamma-independent, and is mediated by the p38 and SAPK/JNK pathway in mitogen-activated protein kinase signaling pathways, which activates NF-kappaB. Our data may give more insights into the different mechanisms contrary to the anti-inflammatory effect of 15d-PGJ(2) on the expression of chemokine genes.     

Inhibition of joint inflammation and destruction induced by anti-type II collagen antibody/lipopolysaccharide (LPS)-induced arthritis in mice due to deletion of macrophage migration inhibitory factor (MIF)

OBJECTIVE: Previous studies have demonstrated that neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibody decreases joint destruction in the collagen-induced arthritis model. The present study was undertaken to investigate whether selective deletion of MIF inhibits inflammation and joint destruction of the anti-type II collagen antibody (anti-CII Ab)/lipopolysaccharide (LPS)-induced arthritis in mice, in order to determine the role of this cytokine in inflammatory arthritis. DESIGN: Anti-CII Ab/LPS-induced arthritis was induced in MIF-deficient and wild-type mice. The effects of anti-MIF polyclonal antibody administration on anti-CII Ab-induced arthritis were also evaluated. RESULTS: The expression of MIF protein and mRNA was induced in anti-CII Ab/LPS-induced arthritis joint tissues. Histopathological arthritis scores for synovial inflammation induced by anti-CII Ab/LPS -induced arthritis were significantly decreased in anti-MIF Ab-treated mice and in MIF-deficient mice compared to wild-type mice. In addition, mRNA levels of MMP-13 and MIP-2 in anti-CII Ab/LPS-induced arthritis joint tissues were significantly reduced in MIF-deficient mice compared to wild-type control mice. CONCLUSIONS: These results indicate that MIF plays a critical role in inflammation and joint destruction in the anti-CII Ab/LPS-induced arthritis model in mice, in part via induction of MMP-13 and neutrophil infiltration through the induction of MIP-2.     

Inhibitory regulation of osteoclast differentiation by interleukin-3 via regulation of c-Fos and Id protein expression

Interleukin-3 (IL-3) is produced under various pathological conditions and is thought to be involved in the pathogenesis of inflammatory diseases; however, its function in bone homeostasis under normal conditions or nature of the downstream molecular targets remains unknown. Here we examined the effect of IL-3 on osteoclast differentiation from mouse and human bone marrow-derived macrophages (BMMs). Although IL-3 can induce osteoclast differentiation of multiple myeloma bone marrow cells, IL-3 greatly inhibited osteoclast differentiation of human BMMs isolated from healthy donors. These inhibitory effects of IL-3 were only observed at early time points (days 0 and 1). IL-3 inhibited the expression of c-Fos and NFATc1 in BMMs treated with RANKL. However, IL-3-mediated inhibition of osteoclast differentiation was not completely reversed by ectopic expression of c-Fos or NFATc1. Importantly, IL-3 induced inhibitor of DNA binding/differentiation (Id)1 in hBMMs, while Id2 were sustained during osteoclast differentiation of mBMMs treated with IL-3. Ectopic expression of NFATc1 in Id2-deficient BMMs completely reversed the inhibitory effect of IL-3 on osteoclast differentiation. Furthermore, inflammation-induced bone erosion was markedly inhibited by IL-3 administration. Taken together, our results suggest that IL-3 plays an inhibitory role in osteoclast differentiation by regulating c-Fos and Ids, and also exerts anti-bone erosion effects.     

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.     

Anti-c-Fms antibody inhibits lipopolysaccharide-induced osteoclastogenesis in vivo

It has been reported that lipopolysaccharide (LPS) has the ability to induce inflammation and osteoclastogenesis. Osteoclast formation is dependent on macrophage-colony-stimulating factor (M-CSF) and ligand for the receptor activator of necrosis factor-kB. In this study, the effect of antibody against c-Fms, which is the receptor of M-CSF, on LPS-mediated osteoclastogenesis was investigated in mice. LPS was administered with or without anti-c-Fms antibody into the supracalvaria of mice. The number of osteoclasts and the levels of mRNA for cathepsin K and tartrate-resistant acid phosphatase, which are osteoclast markers, in mice administered both LPS and anti-c-Fms antibody were lower than those in mice administered LPS alone. The level of tartrate-resistant acid phosphatase 5b as a marker of bone resorption in mice administered both LPS and anti-c-Fms antibody was also lower. Furthermore, the expression of the receptor activator of necrosis factor-kB, which is receptor activator of nuclear factor kappa-B ligand, was increased upon LPS administration, but the expression was inhibited by anti-c-Fms antibody. These results showed that anti-c-Fms antibody inhibits LPS-induced osteoclast formation. In conclusion, M-CSF and its receptor are potential therapeutic targets in bacterial infection-induced osteoclastogenesis, and anti-c-Fms antibody might be useful for inhibition of bacterial infection-induced bone destruction.     

R848, a toll-like receptor 7 agonist, inhibits osteoclast differentiation but not survival or bone-resorbing function of mature osteoclasts

R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-β was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-β to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.     

A novel pathway regulating lipopolysaccharide-induced shock by ST2/T1 via inhibition of Toll-like receptor 4 expression

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-alpha but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-kappaB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-alpha. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.