IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell-cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption...). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) (TNF-alpha)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background  

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.     

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Hepatotoxin rubratoxin B induced the secretion of TNF-alpha, IL-8, and MCP-1 in HL60 cells

Induction of cytokine secretion by rubratoxin B was investigated using HL60 cells. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 were secreted from 40 and 80 microg/ml rubratoxin B-treated cells. In 20 and 40 microg/ml rubratoxin B-treated samples, monocyte chemotactic protein (MCP)-1 was released. These rubratoxin B-induced cytokines are known to promote liver myelocytic cell infiltration, and activate cytokine-recruited cells. As a result, recruited myelocytic cells are considered to contribute to hepatic injury. We investigated the effects of tyrosine kinase inhibitors genistein and emodin. Genistein reduced the release of all three cytokines from rubratoxin B-treated cells. Likewise, emodin diminished the secretion of MCP-1. Alternatively, emodin reversed on the secretion of TNF-alpha, and the release of IL-8 was not affected. Since emodin did not impede rubratoxin B-caused TNF-alpha and IL-8 secretion, they appeared to be regulated differently from MCP-1 secretion, suggesting that rubratoxin B exerts its toxicity using two or more signal transduction pathways.     

RANKL increases the level of Mcl-1 in osteoclasts and reduces bisphosphonate-induced osteoclast apoptosis in vitro

Introduction  

Bisphosphonates are the most widely used class of drug for inhibiting osteoclast-mediated bone loss, but their effectiveness at preventing joint destruction in rheumatoid arthritis has generally been disappointing. We examined whether the ability of bisphosphonates to induce osteoclast apoptosis and inhibit bone resorption in vitro is influenced by the cytokine receptor activator of nuclear factor-kappa B ligand (RANKL), an important mediator of inflammation-induced bone loss.     

Identification and characterization of a sodium/calcium exchanger, NCX-1, in osteoclasts and its role in bone resorption

We provide the first demonstration for a Na+/Ca2+ exchanger, NCX-1, in the osteoclast. We speculate that by using Na+ exchange, NCX-1 couples H+ extrusion with Ca2+ fluxes during bone resorption. Microspectrofluorimetry of fura-2-loaded osteoclasts revealed a rapid and sustained, but reversible, cytosolic Ca2+ elevation upon Na+ withdrawal. This elevation was abolished by the cytosolic introduction (by gentle permeabilization) of a highly specific Na+/Ca2+ exchange inhibitor peptide, XIP, but not its inactive analogue, sXIP. Confocal microscopy revealed intense plasma membrane immunofluorescence with an isoform-specific monoclonal anti-NCX-1 antibody applied to gently permeabilized osteoclasts. Electrophysiological studies using excised outside-in membrane patches showed a low-conductance, Na+-selective, dichlorobenzamil-sensitive, amiloride-insensitive channel that we tentatively assigned as being an NCX. Finally, to examine for physiological relevance, an osteoclast resorption (pit) assay was performed. There was a dramatic reduction of bone resorption following NCX-1 inhibition by dichlorobenzamil and XIP (but not with S-XIP). Together, the results suggest that a functional NCX, likely NCX-1, is involved in the regulation of osteoclast cytosolic Ca2+ and bone resorption.     

Changes in plasma IL-1beta, TNF-alpha and IL-6 after total hip replacement surgery in general or regional anaesthesia

Different anaesthetic methods influence the neuro-immuno-endocrine biologic responses to surgery and may thus possibly interfere with the postoperative course and development of complications. The neuroendocrine system is closely related to the cytokine network. In this study, the effects of general anaesthesia (n=6) and regional spinal/epidural anaesthesia (n=6) on the cytokine response (IL-1beta, TNFalpha, IL-6) to uncemented total hip replacement surgery were evaluated. The postoperative clinical course was uneventful in every case. In both groups, only very low values of plasma IL-beta were measured perioperatively, whereas plasma IL-6 increased postoperatively with peak values 4 h after surgery. The changes in plasma TNF-alpha were not significant. No significant differences in plasma TNF-alpha or IL-6 were found between patients operated in general or in regional anaesthesia. This suggests minor influence of plasma cytokines on the possible beneficial effects of regional anaesthesia on the clinical course after surgery in low risk patients. There were slightly higher TNF-alpha and IL-6 levels after the operation and significantly lower cortisol levels during the operation in the regional anaesthesia group compared to the general anaesthesia group, giving rise to a significant inverse correlation between peak values of IL-6 and peak values of cortisol. This supports the theory that after surgery the inhibitory effect of cortisol on monocyte cytokine production overrides adrenergic stimulation.     

Identification of rat IL-1beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining.

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1beta, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.     

RANKL increases the level of Mcl-1 in osteoclasts and reduces bisphosphonate-induced osteoclast apoptosis in vitro

Introduction

Synovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicans-induced septic arthritis is largely unknown.

Methods

Primary cultures of rat synovial fibroblasts were infected with C. albicans (ATCC90028). Immunocytochemistry, western blotting, and RT-PCR were performed to assess cyclo-oxygenase 2 induction. Phosphorylation of extracellular-regulated kinase (ERK1/2) following infection in the absence or presence of U0126 was assessed by western blotting whilst prostaglandin E2 production was measured by ELISA. Nuclear factor κB (NFκB) translocation was evaluated by an electrophoretic mobility shift assay.

Results

Infection of synovial fibroblasts with C. albicans resulted in cyclo-oxygenase 2 expression and prostaglandin E2 production. Cyclo-oxygenase 2 expression and prostaglandin E2 production was dependent upon extracellular-regulated kinase 1/2 phosphorylation, associated with activation of NFκB and significantly elevated in the presence of laminarin, an inhibitor of dectin-1 activity. Synovial fibroblasts adjacent to C. albicans hyphae aggregates appeared to be the major contributors to the increased levels of cyclo-oxygenase 2 and phosphorylated extracellular-regulated kinase 1/2.

Conclusions

C. albicans infection of synovial fibroblasts in vitro results in upregulation of cyclo-oxygenase 2 and prostaglandin E2 by mechanisms that may involve activation of extracellular-regulated kinase 1/2 and are associated with NFκB activation.     

The modulatory role played by TNF-alpha and IL-1 beta in the inflammatory responses induced by carrageenan in the mouse model of pleurisy

We describe here the modulation caused by intrapleural (i.pl.) injection of the cytokines TNF-alpha and IL-1beta and their specific antibodies in the early (4 h) and late (48 h) inflammatory responses caused by injection of carrageenan (Cg) into the mouse pleural cavity. The antibodies against TNF-alpha and IL-1beta, when injected 30 min prior to Cg, reduced, in a graded and significant manner, both exudation and cell migration in the early (4 h) phase, while they potentiated or had no effect in the late (48 h) phase of Cg response. The natural IL-1 receptor antagonist IL-1RA, given 30 min prior to Cg, reduced the exudation by about 50% and abolished the total and differential cell migration in the early (4 h) and late (48 h) phases of the Cg responses. The i.pl. injection of TNF-alpha or IL-1beta, 5 min prior to Cg, caused graded increase in the exudation of the early (4 h) and late (48 h) phases of the Cg-induced inflammatory responses. In contrast, these treatments markedly reduced the total and differential cell migration at 4 h, while having little or no effect on the late (48 h) phase of the Cg pleurisy. These findings extend previous results and demonstrate that the pro-inflammatory cytokines TNF-alpha and IL-1beta have a critical role in controlling both cell migration and exudation caused by injection of Cg in the mouse pleural cavity. Together, these findings may be relevant to the understanding of the mechanisms involved in airway inflammatory responses.     

Pravastatin immunomodulates IL-6 and C-reactive protein, but not IL-1 and TNF-alpha, in cardio-pulmonary bypass

BACKGROUND: While statins are increasingly used in cardiopulmonary bypass (CPB), the anti-inflammatory effects of individual statins, within the context of various treatment regimes, need further examination. The present study evaluates the anti-inflammatory effectiveness of the short-term, preoperative and intensive postoperative use of pravastatin in CPB. METHOD: Forty three patients undergoing CPB were enrolled in a randomized, prospective clinical study. One group (n = 21), received pravastatin, the other (n = 22) did not. Patients in the pravastatin group received one dose of 40 mg per day for nine days, starting 48 hours before CPB, with an additional dose of 40 mg one hour after surgery. Plasma levels of selected inflammatory mediators were measured at baseline and tracked systematically. RESULTS: Pravastatin reduced postoperative interleukin-6 (IL-6) levels significantly at 24 and 48 hours, and at seven days. Mean +/- SD values, for treated versus untreated patients were: at 24 hours, 159.5 +/- 58.5 versus 251.2 +/- 53.0 pg/mL (p < 0.001); at 48 hours, 81.9 +/- 31.5 versus 194.2 +/- 56.3 pg/mL (p < 0.001); and at seven days, 16.4 +/- 7.2 versus 30.8 +/- 12.6 (p < 0.001). C-reactive protein (CRP) decreased significantly on the seventh postoperative day, when plasma levels were 3.6 +/- 1.1 in the treated patients versus 8.2 +/- 2.1 mg/dL in the controls (p < 0.001). No changes in plasma IL-1 and TNF-alpha were found during entire study. CONCLUSIONS: Pravastatin induced a precocious modulation of IL-6 expression and a later reduction of plasma CRP levels. Pravastatin;s effects on the expression of these pivotal inflammatory mediators strongly support its well-timed use in CPB.     

Overexpression of EXTL3/EXTR1 enhances NF-kappaB activity induced by TNF-alpha

EXTL3/EXTR1 is a member of the EXT gene family, which may represent a class of glycosyltransferases involved in heparan sulfate biosynthesis. It is known that heparan sulfate interacts with a variety of proteins and is therefore implicated in various cellular responses. Here, we examined the effect of EXTL3 on nuclear factor-kappaB (NF-kappaB) activity stimulated by tumor necrosis factor-alpha (TNF-alpha), one of heparin-binding cytokine. The luciferase assay demonstrated that overexpression of EXTL3 enhanced TNF-alpha-induced NF-kappaB activity. This is confirmed with an electrophoretic mobility shift assay. However, EXTL3 did not affect the CD40-mediated NF-kappaB activation. The EXTL3 mutants lacking the amino terminus region failed to enhance the activity. The fluorescence of enhanced green fluorescent protein (EGFP)-fused EXTL3 was observed at the perinuclear region, whereas, the amino terminus-truncated mutant was found in a diffuse cytoplasmic region. These results suggest that EXTL3 may modulate NF-kappaB mediated by TNF-alpha.     

Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1 alpha, and TNF-alpha through nucleotide-binding oligomerization domain 2-mediated signaling in osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and PGE2, LPS and IL-1alpha stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-kappaB ligand (RANKL) or TNF-alpha. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-alpha but not 1alpha,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-alpha in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-alpha-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1alpha, and TNF-alpha through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.     

Recombinant human IL-1 beta and TNF-alpha stimulate production of IL-1 alpha and IL-1 beta by vascular smooth muscle cells and IL-1 alpha by vascular endothelial cells

Vascular endothelial cells (EC) produced IL-1 alpha but not IL-1 beta into extracellular fluids. Vascular smooth muscle cells (SMC), on the other hand, produced both IL-1 alpha and IL-1 beta, and IL-1 beta produced was much higher than IL-1 alpha. The addition of recombinant human IL-1 beta or recombinant human TNF-alpha significantly enhanced IL-1 alpha production in EC, and IL-1 alpha and IL-1 beta production in SMC. IL-1 beta release was not observed even when EC were stimulated with TNF-alpha. These results suggest that the species of released form of IL-1 are different in different cell types and that cytokines enhance IL-1 alpha and IL-1 beta production in SMC and IL-1 alpha production in EC.     

Western blot analysis of bile or intestinal fluid from patients with septic shock or systemic inflammatory response syndrome, using antibodies to TNF-alpha, IL-1 alpha and IL-1 beta

Septic shock or systemic inflammatory response syndrome (SIRS) often develops in patients following burns, traumatic injury, surgery or biliary obstruction. Although the inflammatory cytokines TNF-alpha and IL-1 have been strongly implicated in the development of these syndromes, treatment of patients by the systemic administration of inhibitors of TNF-alpha or IL-1 has shown limited effectiveness. Recent reports suggest that septic shock may be perpetuated by inflammatory cytokines secreted by the liver in response to bacterial translocation resulting from cytokine-induced gastrointestinal damage. The present study sought to demonstrate the presence of high levels of inflammatory cytokines in the bile or small intestine of patients suffering from septic shock or SIRS, with a view to the development of strategies for the reduction of gastrointestinal damage through intraduodenal administration of cytokine inhibitors. Western blot analysis of human bile or intestinal fluid using anti-TNF-alpha antibodies resulted in the detection of a number of bands in samples from patients with septic shock or SIRS. However, these proteins differed antigenically from human recombinant TNF-alpha (rTNF-alpha) and showed no activity in a biological assay for TNF-alpha. Antibodies to IL-1 alpha and IL-1 beta detected several strong bands, some of which appeared to be identical to recombinant IL-1 alpha and IL-1 beta. It is concluded that proteins resembling several known inflammatory cytokines are present in the bile and intestine of septic shock patients, but it is suggested that further work is required to determine the nature and function of these molecules.     

Dendritic cells induced in the presence of GM-CSF and IL-5

Experimental autoimmune encephalomyelitis (EAE) is commonly regarded as an animal model of the human disease multiple sclerosis (MS). Pertussis toxin (PTX) is routinely used for EAE induction in mice. Besides opening the blood-brain barrier, it acts as an adjuvant causing strong expansion of antigen-specific cells after coinjection with neuroantigens in IFA. Using an IL-17 ELISPOT assay we developed previously, we investigated the capability of PTX to induce proteolipid protein peptide 139-151(PLPp)-specific Th-17 cells in the immune periphery and in the thymus after coinjection with PLPp/IFA. PTX was found to induce peripheral PLPp-specific Th-17 cells in the draining lymph node and in the spleen, but not in the thymus. Our study indicates a new mechanism by which microbial agents can initiate or maintain autoimmune reactions and supports the growing role in particular for Th-17 cells in organ-specific autoimmune diseases like multiple sclerosis or EAE.     

The importance of IL-1 beta and TNF-alpha,and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis

Injection of anti-type II collagen Ab and LPS induces arthritis in mice. The levels of IL-1 beta, IL-6, and chemokines (macrophage inflammatory protein (MIP)-1 alpha, MIP-2, and monocyte chemoattractant protein-1) in the hind paws increased with the onset of arthritis and correlated highly with arthritis scores. The level of TNF-alpha was also elevated, but only transiently. Quantitative real-time PCR analysis revealed increases in cytokine and chemokine mRNA. To elucidate the contribution of inflammatory cytokines and chemokines in arthritis development more directly, recombinant proteins, neutralizing Abs, and knockout mice were used. The injection of rIL-1 beta or TNF-alpha, but not IL-6 or chemokines, induced arthritis when mice were i.v. preinjected with anti-type II collagen Ab. However, a single injection of recombinant cytokines or chemokines into the hind paws did not induce swelling. Arthritis development was inhibited by neutralizing Ab against IL-1 beta, TNF-alpha, or MIP-1 alpha. In contrast, the inhibitory effect by anti-MIP-2 Ab was partial and, surprisingly, Abs to IL-6 and monocyte chemoattractant protein-1 showed no inhibitory effect. Furthermore, arthritis development in IL-1R(-/-) mice and TNFR(-/-) mice was not observed at all, but severe arthritis was developed in IL-6(-/-) mice. These results suggest that IL-1 beta and TNF-alpha play more crucial roles than IL-6 or chemokines in this model. Because arthritis was also developed in SCID mice, the development of arthritis in the Ab-induced mice model is due to a mechanism that does not involve T or B cells.     

Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.