Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate

The development of LPS tolerance has been suggested to be mediated by an inhibition of cytokine synthesis. Here we have studied serum IL-6 and TNF levels in mice after LPS administration. Repeated administration of LPS (35 micrograms daily for 4 days) to mice induced a refractoriness (tolerance) to subsequent administrations of LPS in terms of induction of circulating IL-6 and TNF. To investigate the mechanism by which LPS down-regulates its own induction of cytokine synthesis and the relationship between IL-6 and TNF production, we attempted to revert the inhibition of IL-6 and TNF production using agents like PMA or IFN-gamma, previously reported to activate macrophage production of cytokines. Pretreatment with PMA (4 micrograms, 10 min before LPS) partially restored IL-6 production in LPS-tolerant mice given 2 micrograms LPS. On the other hand, PMA did not restore TNF induction in LPS-tolerant mice, even when administered with high doses of LPS (up to 200 micrograms). A similar reversal of LPS resistance to IL-6, but not TNF, induction by PMA was observed in genetically LPS-resistant C3H/HeJ mice. IFN-gamma also restored, although to a lesser extent than PMA, IL-6 production. However, unlike PMA, IFN-gamma could also partially restore TNF production in LPS-tolerant mice, although only when LPS was administered at high doses. By contrast with PMA, IFN-gamma was clearly more active in restoring TNF synthesis than that of IL-6. Similar results were obtained in genetically LPS-unresponsive C3H/HeJ mice. These data suggest that different mechanisms are implicated in the inhibition of IL-6 and TNF synthesis in LPS-tolerant mice and that part of this inhibition can be overcome by PMA or IFN-gamma.     

Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-alpha.

Pretreatment with recombinant human granulocyte CSF (G-CSF) protected mice in two different models of septic shock. Intravenous injection of 250 micrograms/kg G-CSF to mice prevented lethality induced by 5 mg/kg LPS. Injection of 50 micrograms/kg G-CSF protected galactosamine-sensitized mice against LPS-induced hepatitis. In either case, this protection was accompanied by a suppression of LPS-induced serum TNF activity. In contrast, when galactosamine-sensitized mice were pretreated with 50 micrograms/kg murine recombinant granulocyte/macrophage CSF instead of G-CSF and subsequently challenged with LPS, serum TNF activity was significantly enhanced and mortality was increased. The suppressive effect of G-CSF on LPS-induced TNF production was also demonstrated in rats. In vivo, no TNF was detectable in the blood of LPS-treated rats, which had been pretreated with G-CSF. Ex vivo, alveolar macrophages, bone marrow macrophages, Kupffer cells, or peritoneal macrophages prepared from G-CSF-treated rats produced significantly less TNF upon stimulation with LPS than corresponding populations from control rats. However, when these macrophage populations were incubated with G-CSF in vitro, LPS-induced TNF production was unaffected. These data suggest that the G-CSF-mediated suppression of TNF production is not a direct effect of G-CSF on macrophages. To examine whether, independent of the protection against LPS, G-CSF treatment still activated neutrophils, it was demonstrated that granulocytes from G-CSF-treated rats were primed for PMA-induced oxidative burst and for ionophore/arachidonic acid-stimulated lipoxygenase product formation. The experiments of this study support the notion that G-CSF is a negative feedback signal for macrophage-derived TNF-alpha production during Gram-negative sepsis.     

N-acetylcysteine and glutathione as inhibitors of tumor necrosis factor production.

TNF is a major mediator in the pathogenesis of endotoxic shock, and its inhibition has a protective effect in various animal models of sepsis or endotoxin (lipopolysaccharide, LPS) toxicity. LPS treatment also induces an oxidative damage mediated by increased production of reactive oxygen intermediates. N-Acetylcysteine (NAC) is an antioxidant and a precursor of the synthesis of glutathione (GSH) and was reported to protect against LPS toxicity and LPS-induced pulmonary edema. In this study we investigated the effect of NAC on TNF production and LPS lethality in mice. The results indicated that oral administration of NAC protects against LPS toxicity and inhibits the increase in serum TNF levels in LPS-treated mice. The inhibition was not confined to the released form of TNF, since NAC also inhibited LPS-induced spleen-associated TNF. On the other hand, the inhibitor of GSH synthesis, DL-buthionine-(SR)-sulfoximine (BSO), had the opposite effect of potentiating LPS-induced TNF production, and this was associated with a decrease in liver GSH levels. Repletion of liver GSH with NAC reversed this effect. NAC was also active in inhibiting TNF production and hepatotoxicity in mice treated with LPS in association with a sensitizing dose of Actinomycin D. These data indicate that GSH can be an endogenous modulator of TNF production in vivo. On the other hand, NAC pretreatment did not inhibit other effects of LPS, particularly induction of serum IL-6, spleen IL-1 alpha, and corticosterone, in the same experimental model, suggesting that the observed effect could be specific for TNF.     

Pretreatment with granulocyte-colony stimulating factor decreases lipopolysaccharide-induced interferon-gamma production in mice in association with the production of interleukin-18

We studied the effects of pretreatment with granulocyte-colony stimulating factor (G-CSF) on the production of pro- and anti-inflammatory cytokines induced by lipopolysaccharide (LPS). Mice received G-CSF or control saline once a day for 7 days or once at 1 h before the injection of LPS. Cytokines were measured by enzyme-linked immunosorbent assay or antibody-based electrochemiluminescence assay and cytokine mRNA was measured by RNAse protection assay. Mice pretreated with G-CSF for 7 days before LPS had lower serum levels of LPS-induced interferon-gamma (IFN-gamma) and higher levels of interleukin (IL)-6 and IL-10 than controls. G-CSF-pretreated mice also had lower mRNA levels of IFN-gamma and higher mRNA levels of IL-6 and IL-10 in the spleen and/or liver than controls. G-CSF-pretreated mice had serum levels of tumor necrosis factor, IL-12 p70 and IL-12 p40 similar to controls. G-CSF-pretreated mice had lower levels of spleen IL-18 than controls-serum IL-18 being undetectable in mice after LPS-and lower levels of IL-18 mRNA in the spleen. Mice pretreated with G-CSF 1 h before LPS had lower levels of serum IFN-gamma and spleen IL-18 than controls. G-CSF pretreatment alters the expression of LPS-induced cytokines with a decrease in pro-inflammatory IFN-gamma and an increase in anti-inflammatory IL-6 and IL-10. G-CSF decrease of IL-18 production may be a major mechanism explaining the effects of G-CSF on the production of IFN-gamma.     

clc is co-expressed with clf or cntfr in developing mouse muscles

BACKGROUND: The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor alpha component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR). RESULTS: Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR. CONCLUSION: This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development.     

Lps induces IL-6 in the brain and in serum largely through TNF production

We investigated the relative contribution of IL-6 and PGE2 directly induced by LPS and indirectly induced via TNF, using in vivo and in vitro models in the mouse. In these models we have used as tools an anti-TNF antibody and a cyclooxygenase inhibitor, the S enantiomer of ketoprofen (S-KPF). Anti-TNF antibodies inhibited LPS-induced IL-6 production in three different models: IL-6 production by mouse peritoneal macrophages in vitro; serum IL-6 levels induced by intraperitoneal LPS; and brain IL-6 levels induced by an intracerebroventricular injection of LPS. However, in vitro anti-TNF antibodies, did not inhibit LPS-induced PGE2, indicating that this effect is not mediated by TNF. Since PGE2 has an opposite effect on TNF and IL-6 production, inhibiting that of TNF but inducing that of IL-6, we investigated the effect of S-KPF on TNF and IL-6 production in vivo following LPS injection. Both TNF and IL-6 induction was augmented by S-KPF, but anti-TNF antibodies abolished the augmentation of IL-6 production. Thus, the effect of anti-inflammatory drugs on IL-6 production in some models can be secondary to their effect on TNF production.     

Induction of interferons (IFNs) and tumor necrosis factor (TNF) in mice by a novel glycolipid trehalose 2,3,6'-trimycolate from Rhodococcus aurantiacus (Gordona aurantiaca).

The immunomodifying activity of a novel mycoloyl glycolipid, trehalose 2,3,6'-trimycolate (GaGM), from a unique psychrophilic acid-fast bacterium, Rhodococcus aurantiacus, was examined. ICR mice were primed intravenously (i.v.) or intraperitoneally (i.p.) with liposomes containing GaGM (300 micrograms/mouse), and were administered LPS dissolved in saline (25 micrograms/mouse, i.v.) 2 weeks later. Two hours after injection of LPS, interferons (IFNs) and tumor necrosis factor (TNF) were induced significantly in mice sera. The increase in activities of IFNs and TNF was approximately paralleled with granuloma formation in spleen of mice primed with GaGM. However, IFNs and TNF were not induced either in mice primed with GaGM but not elicited with LPS, or in those primed with GaGM and elicited by GaGM. Both activities induced were lower in mice primed with trehalose mono- or dimycolate from R. aurantiacus (GaTMM, GaTDM) or TDM from Nocardia rubra than in GaGM-primed mice. Time course study showed that the maximum activity of each interferon (alpha, beta, or gamma) was observed at different stages after LPS administration; IFN-alpha, IFN-beta, and IFN-gamma appeared 3, 2, and 6 hours most abundantly after LPS administration, respectively.     

Effects of IL-18 and IL-10 pre-treatment on the alteration of endogenous cytokines in liver and spleen of mice with experimental endotoxemia

Mechanisms of interleukin-18 (IL-18) and interleukin-10 (IL-10) in lipopolysaccharide (LPS) induced endotoxemia are not clear; their protective role is being investigated so that they may effectively modulate the host cytokine levels during endotoxemia. The aim of the study was to evaluate protective effects of IL-18 and IL-10 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury was determined by estimation of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), serum nitric oxide (NOx), hepatic anti-oxidant enzyme and cytokine content in LPS (250 microg/kg) induced endotoxemic mice receiving either IL-18 (500 ng/mouse) or IL-10 (600 ng/mouse) treatment. Mice (87% of IL-10 treated and 74% of IL-18 treated) survived when administered prior to LPS challenge. Pre-treatment of mice with either IL-10 or IL-18 followed by LPS, lead to reduction in SGPT and SGOT level, serum NOx, and altered hepatic anti-oxidant enzymes activity and myeloperoxidase activity than the only LPS treated group. Marked reduction in the amounts of LPS-induced hepatic and splenic TNF-u content has been observed after IL-10 pre-treatment. Results suggested that attenuating the induction of TNF-alpha and IFN-gamma and subsequent induction of nitric oxide formation in response to LPS may in part account for efficient protection by IL-18 and IL-10 in the reduction of LPS-induced liver injury.     

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.     

Solution structure of the C-terminal domain of the ciliary neurotrophic factor (CNTF) receptor and ligand free associations among components of the CNTF receptor complex

The functional receptor complex of ciliary neurotrophic factor (CNTF), a member of the gp130 family of cytokines, is composed of CNTF, the CNTF receptor alpha (CNTFR), gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature of the receptor-mediated interactions in this complex has not yet been resolved. To address this issue we have determined the solution structure of the C-terminal or BC domain of CNTFR and studied the interactions of CNTFR with LIFR and gp130. We reported previously that the membrane distal cytokine-binding domain (CBD1) of LIFR could interact in vitro with soluble CNTFR (sCNTFR) in the absence of CNTF. Here we show that the CBD of human gp130 can also bind in vitro to sCNTFR in the absence of CNTF. In addition, the gp130 CBD could compete with the LIFR CBD1 for the binding of sCNTFR. Substitution of residues in the gp130 CBD, the LIFR CBD1, and the CNTFR BC domain that are expected to be involved in receptor-receptor interactions significantly reduced their interactions. An NMR chemical shift perturbation study of the interaction between the BC domains of CNTFR and gp130 further mapped the interaction surface. These data suggest that both the gp130 CBD and the LIFR CBD1 interact with CNTFR in a similar way and provide insights into the nature of the CNTF receptor complex.     

High serum IL-6 level reflects susceptible status of the host to endotoxin and IL-1/tumor necrosis factor.

Patients with high level of serum endotoxin did not necessarily develop into lethal shock, whereas some patients died of septic shock even when their serum endotoxin levels were low. These results indicate that limiting factor which determines the host to be endotoxin shock principally depends on the host susceptibility to endotoxin instead of serum endotoxin level. To understand this susceptible status of the host to endotoxin, we used Propionibacterium acnes primed mouse endotoxin shock model. We found that P. acnes-primed mice responded to low dose of LPS by enhanced production of IL-1 and TNF. And such mice were highly susceptible to the lethal shock inducing effect of IL-1 and/or TNF, which also induced high level of serum IL-6 in these mice. Therefore, measurement of serum IL-6 level provides us with the information of the preceding exposure of the host to either LPS or IL-1 and/or TNF and the highly susceptible status of the host to these stimuli. Based on these results obtained from animal model, we investigated the relationship between serum IL-6 levels and serum endotoxin levels in the patients with malignant hematologic disorders. We found that these patients fell into two groups; an endotoxin susceptible group, equivalent to P. acnes-primed mice, showing high level of serum IL-6 with low level of serum endotoxin, and a nonendotoxin susceptible group, equivalent to P. acnes-nonprimed mice, showing low or undetectable level of serum IL-6 with high level of serum endotoxin. We propose that the measurement of serum IL-6 level in the patients positive for endotoxin is a useful tool in evaluating diagnosis and prognosis of endotoxin shock.     

Taurolidine, an analogue of the amino acid taurine, suppresses interleukin 1 and tumor necrosis factor synthesis in human peripheral blood mononuclear cells.

Taurolidine (Geistlich Pharm, AG, Wolhusen, Switzerland), a derivative of the amino acid taurine, is commonly used in some parts of the world as an adjunctive therapy for various infections. Its mechanism of action is thought to be related to its antimicrobial properties, including its ability to interfere with some of the biological activities of endotoxin (lipopolysaccharide, LPS). For example, taurolidine has been shown to protect animals against endotoxic shock and death. In this study we examined the ability of taurolidine to block LPS-induced tumor necrosis factor (TNF) and interleukin 1 (IL-1) synthesis in human peripheral blood mononuclear cells (PBMC) from 27 donors. We observed a dose-dependent reduction in the synthesis of these two cytokines when taurolidine was preincubated with LPS before being added to PBMC. This reduction was independent of the molar ratio of taurolidine to LPS but was related to the concentration of taurolidine present in the PBMC cultures. There was a 80 to 90% reduction in total IL-1 and TNF synthesis induced by LPS at concentrations of taurolidine of 40 to 100 micrograms/mL; the vehicle was without effect. Following a 30-min preincubation with PBMC, taurolidine could be washed from the cells and still suppress cytokine synthesis induced by LPS. Using release of lactic acid dehydrogenase, 100 micrograms/mL of taurolidine was not toxic for PBMC. Taurolidine also reduced IL-1 and TNF synthesis induced by the Staphylococcus aureus-derived toxic shock syndrome toxin-1 as well as that induced by nontoxic heat-killed Staphylococcus epidermidis organisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of proinflammatory cytokines on lipopolysaccharide-induced phase shifts in locomotor activity circadian rhythm

We previously reported that early night peripheral bacterial lipopolysaccharide (LPS) injection produces phase delays in the circadian rhythm of locomotor activity in mice. We now assess the effects of proinflammatory cytokines on circadian physiology, including their role in LPS-induced phase shifts. First, we investigated whether differential systemic induction of classic proinflammatory cytokines could explain the time-specific behavioral effects of peripheral LPS. Induction levels for plasma interleukin (IL)-1α, IL-1β, IL-6, or tumor necrosis factor (TNF)-α did not differ between animals receiving a LPS challenge in the early day or early night. We next tested the in vivo effects of central proinflammatory cytokines on circadian physiology. We found that intracerebroventricular (i.c.v.) delivery of TNF-α or interleukin IL-1β induced phase delays on wheel-running activity rhythms. Furthermore, we analyzed if these cytokines mediate the LPS-induced phase shifts and found that i.c.v. administration of soluble TNF-α receptor (but not an IL-1β antagonistic) prior to LPS stimulation inhibited the phase delays. Our work suggests that the suprachiasmatic nucleus (SCN) responds to central proinflammatory cytokines in vivo, producing phase shifts in locomotor activity rhythms. Moreover, we show that the LPS-induced phase delays are mediated through the action of TNF-α at the central level, and that systemic induction of proinflammatory cytokines might be necessary, but not sufficient, for this behavioral outcome.     

Suppressive activity of a macrolide antibiotic, roxithromycin, on pro-inflammatory cytokine production in vitro and in vivo

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro-inflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL-1beta and TNF-alpha contents by enzyme-linked immunosorbent assay. RXM suppressed the in vitro production of IL-1beta and TNF-alpha in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 microg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL-1beta and TNF-alpha in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5-12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL-1beta and TNF-alpha in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.     

Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.     

Endotoxin rapidly induces changes in lipid metabolism that produce hypertriglyceridemia: low doses stimulate hepatic triglyceride production while high doses inhibit clearance.

Hyperlipidemia frequently accompanies infectious diseases and may be due to increases in lipoprotein production or decreases in lipoprotein clearance. The administration of endotoxin (LPS) has been used to mimic infection and prior studies demonstrate that LPS produces hypertriglyceridemia. In the present study in rodents, the dose of LPS necessary to induce hyperlipidemia was orders of magnitude less than that necessary to induce shock and death. As little as 10 ng/100 g body weight induced hypertriglyceridemia and this increase in serum triglyceride levels occurred rapidly (78% increase at 2 h). At high doses of LPS (50 micrograms/100 g body weight), the clearance of triglyceride-rich lipoproteins was decreased. At low doses of LPS (100 ng/100 g body weight), triglyceride clearance was not altered but the hepatic secretion of triglyceride was increased. Low dose LPS stimulated hepatic de novo fatty acid synthesis and lipolysis, both of which provided a source of fatty acids for the increase in hepatic triglyceride production. High dose LPS did not increase hepatic fatty acid synthesis or peripheral lipolysis, and hepatic triglyceride secretion was not stimulated. Thus, low dose LPS produces hypertriglyceridemia by increasing hepatic lipoprotein production, while high dose LPS produces hypertriglyceridemia by decreasing lipoprotein catabolism. Administration of anti-tumor necrosis factor (TNF) antibodies or interleukin 1 (IL-1) receptor antagonist did not prevent the increase in serum triglyceride levels induced by LPS. However, anti-TNF antibodies and interleukin 1 receptor antagonist (IL-1ra) blocked the increase in serum triglycerides induced by TNF or IL-1, respectively. These data suggest that neither of these cytokines is absolutely required for the increase in serum triglycerides induced by LPS, raising the possibility that other cytokines, small molecular mediators, or LPS itself may play a crucial role.     

Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-alpha, IL-1, and RANKL.

Lipopolysaccharide (LPS), a cell component of Gram-negative bacteria, is a pathogen of inflammatory bone loss. To examine the effects of LPS on the survival and fusion of osteoclasts, mononuclear osteoclasts (preosteoclasts, pOCs) were collected from a mouse co-culture system and cultured in the presence or absence of LPS. Most pOCs died within 24 h in the absence of any stimulus. LPS as well as receptor activator of NF-kappaB ligand (RANKL) supported the survival of pOCs, and induced their fusion to form multinucleated cells (MNCs). Like authentic osteoclasts, MNCs induced by LPS expressed calcitonin receptors, and formed actin rings on culture plates. LPS-induced MNC formation in pOC cultures was observed even in the presence of osteoprotegerin and interleukin (IL)-1-receptor antagonists. MNC formation was also stimulated by LPS in pOC cultures prepared from tumor necrosis factor (TNF)-receptor-I or TNF-receptor-II deficient mice. LPS induced the degradation of IkappaB in pOCs within 20 min. Lactacystin, an inhibitor of NF-kappaB activation, and wortmannin, an inhibitor of phosphatidylinositol-3 kinase, strongly inhibited LPS-induced MNC formation in pOC cultures. LPS induced pit-forming activity of pOCs in the presence of macrophage-colony stimulating factor (M-CSF). These findings suggest that LPS stimulates the survival and fusion of pOCs, independent of RANKL, IL-1 or TNF-alpha action. Activation of NF-kappaB and phosphatidylinositol-3 kinase appeared to be involved in LPS-induced effects on pOCs. These observations suggest that LPS is involved directly in inflammatory bone loss, and also indirectly through the production of LPS-induced host factors such as IL-1 and TNF-alpha.