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PubMed2005

The role of postischemic reperfusion injury and other nonantigen-dependent inflammatory pathways in transplantation.

The Injury Hypothesis, first published in 1994 and modified several times between 1996 and 2002, holds that the reactive oxygen species-mediated reperfusion injury to allografts initiates and induces the alloimmune response and contributes to alloatherogenesis. Recent experimental and clinical evidence in support of the concept is presented suggesting that (1) reactive oxygen species-mediated allograft injury activates the innate immune system of the donor and recipient; (2) injury-induced putative endogenous ligands of Toll-like receptors (TLRs) of host origin such as heat shock proteins interact with and activate TLR4-bearing dendritic cells that mature and induce the adaptive alloimmune response (acute rejection), and interact with and activate TLR4-bearing vascular cells contributing to the development of alloatherosclerosis (chronic rejection); and (3) TLR4-triggered signaling, involved in the establishment of a reperfusion injury, seems to use myeloid differentiation marker 88-independent, Toll/interleukin-1 receptor domain containing adaptor inducing interferon-beta-dependent pathways that are associated with the maturation of dendritic cells and induction of interferon-inducible genes.

Animals↗
PubMed2004

Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4.

Chlamydophila pneumoniae, an obligately intracellular Gram-negative bacterium and a common causative agent of respiratory tract infections, has been implicated in the induction and progression of atherosclerosis and coronary artery disease. In this study, the signalling mechanism of C. pneumoniae in human fibroblasts, a prominent cell population in chronic inflammation and persistent infection, contributing to plaque formation, was investigated. C. pneumoniae elementary bodies were demonstrated to up-regulate the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) in human fibroblasts. The effect was independent of the chlamydial lipopolysaccharide and was likely to be mediated by a heat-labile chlamydial protein. Furthermore, an anti-Toll-like receptor 4 (TLR4) antibody was shown to abolish C. pneumoniae-induced cell activation, whereas an anti-TLR2 antibody had no effect, indicating the role of TLR4 in p44/p42 MAPK activation. Ca2+/calmodulin-dependent protein kinase inhibitor KN-62 and phosphodiesterase 4 (PDE 4) inhibitor Rolipram enhanced C. pneumoniae-induced MAPK phosphorylation and attenuated C. pneumoniae infectivity in vitro. Together the results indicate that C. pneumoniae triggers rapid TLR4-mediated p44/p42 MAPK activation in human fibroblasts and chemical enhancement of MAPK phosphorylation modulates in vitro infection at the molecular level.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2006

Phenol/water extract of Treponema socranskii subsp. socranskii as an antagonist of Toll-like receptor 4 signalling.

Treponema socranskii is one of the most frequently found oral spirochaetes in periodontitis and endodontic infections. LPS or glycolipids from bacteria are potent stimulators of innate immune and inflammatory systems. In this study the bioactivity of a phenol/water extract from T. socranskii subsp. socranskii (TSS-P) was analysed. TSS-P showed minimal endotoxicity and no inducing potential for proinflammatory cytokines (TNF-alpha and IL-8) or for intercellular adhesion molecule-1 (ICAM-1) in human monocyte cell line THP-1 cells and primary cultured human gingival fibroblasts. Rather, it inhibited ICAM-1 expression and IL-8 secretion from cells stimulated by the LPS of Escherichia coli and Actinobacillus actinomycetemcomitans, which are known to be Toll-like receptor 4 (TLR4) agonists. However, this antagonistic activity was not shown in cells stimulated by peptidoglycan or IL-1beta. As its antagonistic mechanism, TSS-P blocked the binding of E. coli LPS to LPS-binding protein (LBP) and CD14, which are molecules involved in the recruitment of LPS to the cell membrane receptor complex TLR4-MD-2 for the intracellular signalling of LPS. TSS-P itself did not bind to MD-2 or THP-1 cells, but inhibited the binding of E. coli LPS to MD-2 or to the cells in the presence of serum (which could be replaced by recombinant human LBP and recombinant human CD14). The results suggest that TSS-P acts as an antagonist of TLR4 signalling by interfering with the functioning of LBP/CD14.

Antigens, Surface↗
PubMed2005

Expression of tumour necrosis factor receptor and Toll-like receptor 2 and 4 on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating.

Toll-like receptors (TLRs) are involved in the recognition of bacterial products and thus participate in the induction of the inflammatory cascade. However, much less is known about the evolution of leucocyte TLR expression during human inflammatory stress. We hypothesized that a decrease in leucocyte TLRs could account for the so-called tolerance or hyporesponsiveness state to subsequent stimulation with bacteria-derived products. Because of the profound monocytopenia that ensues after in vivo lipopolysaccharide (LPS) challenge, we also compared monocyte TLR expression using two different techniques of flow cytometric gating. In a first set of experiments, 17 healthy volunteers underwent LPS challenge. Blood was drawn at different time-points and analysed by flow cytometry using light scatter gating and one-colour analysis to assess the expression of the tumour necrosis factor receptor (TNFR) and TLR2 and TLR4 on both monocytes and granulocytes. In a second set of experiments, the assessment of those receptors was made using a more specific gating method that utilized light scatter and CD14 immunofluorescence in a two-colour analysis. This was performed using whole blood drawn from five healthy volunteers and incubated ex vivo for different time periods with or without LPS and in 12 volunteers who underwent LPS challenge in vivo. The pattern of expression for monocyte TNFR was similar for both types of gating. Using only the light scatter gating, an initial drop of TLR 2 and 4 was observed on monocytes. By contrast, when using light scatter x immunofluorescence gating, an up-regulation of these two receptors following both in vivo and in vitro LPS exposure was observed. LPS up-regulates the expression of TLRs on monocytes and granulocytes. Depending upon the methodology utilized, contrasting results were obtained with respect to TLR2 and TLR4 expression. The flow cytometric gating technique used is of importance in determining cellular TLR2 and TLR4 expression, especially in blood samples exhibiting significant monocytopenia.

Adolescent↗
PubMed2006

Expression of Toll-like receptors and their association with cytokine responses in peripheral blood mononuclear cells of children with acute rotavirus diarrhoea.

To understand virus and host interactions and host responses to rotavirus infection in children, we analysed by real-time polymerase chain reaction (PCR) the expression of mRNA for five Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR7 and TLR8) and four T helper (Th)1 and Th2 cytokines [interleukin (IL)-2, IL-12, interferon (IFN)-gamma and IL-4) in peripheral blood mononuclear cells (PBMC) of children with acute rotavirus diarrhoea. We observed significantly higher expression of genes encoding TLR2, TLR3, TLR4, TLR7 and TLR8 in PBMC of 41% (31/75) patients within 3 days of illness onset than those in healthy children. After 3 days of illness onset, only TLR3 and TLR8 mRNA expressions were still significantly (P<0.05) increased in 59% (44/75) children with diarrhoea. We also observed significantly (P<0.05) elevated expression of IL-12p40 and IFN-gamma in PBMC of patients during the entire period of illness and the first 3 days of illness, respectively. We further demonstrated a weak but significant association between elevated levels of gene expression of four TLRs (TLR2, TLR3, TLR4 and TLR8) and IFN-gamma. Our results suggest that multiple TLRs may modulate the immune response in the acute phase of rotavirus infection and play a role in the activation of IFN-gamma.

Acute Disease↗
PubMed2004

Toll-like receptor-4 Asp299Gly polymorphism does not influence progression of atherosclerosis in patients with familial hypercholesterolaemia.

BACKGROUND: Toll-like receptor-4 (TLR4) is a major receptor for inflammatory stimuli potentially involved in the pathogenesis of atherosclerosis, such as lipopolysaccharide (LPS) and heat-shock proteins. The Asp299Gly polymorphism of the TLR4 gene has been associated with a reduced intima-media thickness (IMT) of the common carotid artery in healthy individuals. We have investigated whether the presence of the Asp299Gly polymorphism in patients with familial hypercholesterolaemia (FH) has a similar protective effect, and whether it influences the effects of HMG-CoA reductase treatment. MATERIALS AND METHODS: A cohort of 293 FH patients and 200 healthy volunteers were genotyped for the presence of the Asp299Gly allele using polymerase chain reaction followed by restriction fragment length polymorphism analysis. Intima-media thickness measurements, inflammatory parameters and the effect of HMG-CoA reductase inhibitors were compared between the patients with and without Asp299Gly allele. RESULTS: The Asp299Gly allele was present in 10.6% of the FH patients and 11.0% of the healthy individuals. Whereas the FH patients carrying the Asp299Gly allele displayed a reduced absolute IMT value compared with the FH patients carrying the wild-type allelle, the difference did not reach statistical significance. In addition, the effect of treatment with HMG-CoA reductase inhibitors was not influenced by the presence of Asp299Gly allele. CONCLUSION: The presence of the Asp299Gly allele of the TLR4 gene does not seem to exert a major influence on the progression of atherosclerosis in patients with FH.

Adult↗
PubMed2004

Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components.

Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-kappaB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Mechanism of up-regulation of immunoglobulin A production in the intestine of mice unresponsive to lipopolysaccharide.

The mechanisms by which immunoglobulin A (IgA) production up-regulates in the intestine of Toll-like receptor-4 (TLR4)-mutated mice were investigated. When TLR4-mutated, C3H/HeJ and BALB/lps(d) mice received oral administration of cholera toxin (CT), not only CT-specific IgA levels in the intestinal lavage but also the number of IgA-producing cells in intestinal lamina propria (iLP) significantly increased compared with those of the wild-type C3H/He and BALB/c mice. Interleukin (IL)-5-producing cells and CD86+ cells in iLP also significantly increased in C3H/HeJ mice. The expression of major histocompatibility complex class II and CD86 on cells present in Peyer's patches (PPs) of C3H/HeJ mice was higher than those of C3H/He mice. In non-immunized C3H/HeJ mice, the expression of transforming growth factor-beta (TGF-beta) mRNA and the percentages of IL-10-producing cells in PPs but not in spleen increased when compared with those in C3H/He mice. The suppressor of cytokine signalling-1 (SOCS-1) was expressed in PPs of C3H/He mice but not C3H/HeJ mice. These results indicate that high IgA levels in the intestine of TLR4-mutated mice are due to up-regulation of TGF-beta and IL-10 and the lack of regulation by SOCS-1.

Animals↗
PubMed2003

Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice.

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Responses to free lipopolysaccharide and Escherichia coli by normal human intestinal macrophages, following their migration out of the lamina propria.

Intestinal macrophage responses to luminal bacteria and their constituents are important in mucosal inflammatory responses. We investigated the responses of intestinal macrophages to free lipopolysaccharide (LPS) and Escherichia coli. Macrophages were isolated from normal terminal ileum and colon by allowing them to migrate out of the lamina propria of mucosal samples denuded of epithelial cells. Following exposure to free LPS or fluorescein-labelled E. coli, responsiveness was studied by intracellular expression of tumour necrosis factor-alpha (TNF-alpha). CD14, CD33, CD68, TLR2 and TLR4 expression was studied by fluorescence-activated cell sorter (FACS). TLR and NOD2 expression was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). CD14 was expressed by 36.5 +/- 4.0% of the macrophages obtained following migration out of the lamina propria. These cells also expressed TLR2, TLR4 and NOD2. Of cells exposed to free LPS or those that had taken up E. coli, a greater proportion of CD14(+) than CD14(-) macrophages expressed intracellular TNF-alpha. Moreover, a greater proportion of macrophages (CD14(+) and CD14(-)) demonstrated responses to E. coli than free LPS. In conclusion, a proportion of macrophages obtained following migration out of the lamina propria of normal terminal ileal and colonic mucosal samples express CD14, TLR2 and TLR4. These cells respond to free LPS and E. coli, as demonstrated by the expression of TNF-alpha.

Aged↗
PubMed2006

Hypoallergenic fragment of Par j 2 increases functional expression of Toll-like receptors in atopic children.

BACKGROUND: Parietaria judaica (Par j) is one of the main causes of allergy in the Mediterranean countries. The activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) inhibits nasal inflammation of atopic children. OBJECTIVE: To examine, in vivo and in vitro, the effect of recombinant Par j 2 (rPar j 2) and of its fragments (1-55 and 52-102) on atopic children. METHODS: We used skin prick test for in vivo evaluations. We assessed, in vitro, in peripheral blood mononuclear cells (PBMC), the effect of rPar j 2 and of the two fragments on neutrophil chemotaxis, on CD45RO, on TLR2 and TLR4 expression, on LPS binding and on interferon (IFN)-gamma release, by a microchemotaxis chamber, by flow cytometry and by enzyme-linked immunosorbent assay, respectively. RESULTS: In vivo while rPar j 2 induced a positive skin reaction, 1-55 and 52-102 fragments did not. In vitro, while rPar j 2 increased both CD45RO expression and neutrophils chemotaxis in PBMC, both Par j 2 fragments did not. 1-55 fragment of Par j 2 upregulated both TLR2 and TLR4 expression and LPS binding, while the rPar j 2 and 52-102 fragment did not. Finally, 1-55 fragment of Par j 2 induced IFNgamma release, while the rPar j 2 and 52-102 fragment did not. CONCLUSIONS: Hypoallergenic 1-55 fragment, upregulating innate immunity receptors and increasing IFNgamma, might re-orientate, in atopics, the immune system toward a physiologic balance between Th1 and Th2 responses.

Adolescent↗
PubMed2005

Effects of 15-deoxy-Delta12,14-prostaglandin J2 on the expression of Toll-like receptor 4 and 2 in the murine lung in the presence of lipopolysaccharide.

1. Previously, we have demonstrated that 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) enhances acute lung injury induced by lipopolysaccharide (LPS) in mice. The enhancement in acute lung injury by 15d-PGJ2 was concomitant with the enhanced expression of several pro-inflammatory cytokines in the lung. However, other underlying mechanisms of this enhancement remain to be elucidated. The present study investigated the effects of 15d-PGJ2 on the expression of Toll-like receptor (TLR) 4 and 2 in the lung in the absence or presence of LPS. 2. In the present study, ICR mice were divided into four experimental groups that received (intratracheally) vehicle, LPS (125 microg/kg), 15d-PGJ2 (1 mg/kg) or 15d-PGJ2 + LPS. The mRNA expression of both TLR4 and 2 in the lung was evaluated 4 h after intratracheal administration. 3. 15-Deoxy-Delta12,14-prostaglandin J2 enhanced the mRNA expression of both TLR4 and 2 in the presence of LPS. 4. These results suggest that the enhancing effects of 15d-PGJ2 on LPS-induced acute lung injury may be explained, at least in part, by its effect on the lung expression of TLR4 and 2.

Animals↗
PubMed2005

Evidence of Toll-like receptor molecules on human platelets.

Platelets are primarily involved in thrombosis and haemostasis, and they have recently been shown to have a role in innate immunity and in inflammation. We have determined the markers of innate immunity that are expressed by platelets, specifically the Toll-like receptors (TLR), originating from mixes of platelet concentrates (MPC, n = 5) between day zero and day five after blood collection. The surface membrane and intracellular expression of TLR were measured, both after and without permeabilization, using flow cytometry. We observed weak expression of TLR2, TLR4 and TLR9 on the surface of CD41(+) platelets. The expression levels of TLR4 were high (59 +/- 2.2%). Moreover, there was a significant expression of TLR2 (47.5 +/- 4.8%), TLR4 (78.8 +/- 1.3%) and TLR9 (34.2 +/- 7.5%) in the cytoplasm of CD41(+) platelets. The expression of the three receptors did not change significantly during the course of the 5 day observation period. The percentage of TLR expression is significantly modulated between activated versus non-activated platelets, both after and without permeabilization (P < 0.01). Study of the expression of TLR could increase our knowledge of the level of platelet participation during an immune reaction and inflammation. In the same way as the platelet ligand/receptor pair CD40L/CD40 is, the TLR are expressed by platelets, and could serve as a link between innate and adaptive immunity.

Blood Platelets↗
PubMed2005

Antimicrobial peptides and endotoxin inhibit cytokine and nitric oxide release but amplify respiratory burst response in human and murine macrophages.

Antimicrobial peptides (AMPs), in addition to their antibacterial properties, are also chemotactic and signalling molecules that connect the innate and adaptive immune responses. The role of AMP [alpha defensins, LL-37, a cathepsin G-derived peptide (CG117-136), protegrins (PG-1), polymyxin B (PMX) and LLP1] in modulating the respiratory burst response in human and murine macrophages in the presence of bacterial endotoxin [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] was investigated. AMP were found to neutralize endotoxin induction of nitric oxide and TNFalpha release in macrophages in a dose-dependent manner. In contrast, macrophages primed overnight with AMP and LOS or LPS significantly enhanced reactive oxygen species (ROS) release compared with cells primed with endotoxin or AMP alone, while no responses were seen in unprimed cells. This enhanced ROS release by macrophages was seen in all cell lines including those obtained from C3H/HeJ (TLR4-/-) mice. Similar effects were also seen when AMP and endotoxin were added directly with zymosan to trigger phagocytosis and the respiratory burst in unprimed RAW 264.7 and C3H/HeJ macrophages. Amplification of ROS release was also demonstrated in a cell-free system of xanthine and xanthine oxidase. Although AMP inhibited cytokine and nitric oxide induction by endotoxin in a TLR4-dependent manner, AMP and endotoxin amplified ROS release in a TLR4-independent manner possibly by exerting a prolonged catalytic effect on the ROS generating enzymes such as the NADPH-oxidase complex.

Animals↗
PubMed2006

Non-lipooligosaccharide-mediated signalling via Toll-like receptor 4 causes fatal meningococcal sepsis in a mouse model.

Meningococcal lipooligosaccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis with disease severity correlating with circulating concentrations of LOS and proinflammatory cytokines. In this study we show that the proinflammatory response to live meningococci in a mouse model of sepsis involves TLR4, but can develop independently of the expression of LOS. This is supported by data showing that in vivo an isogenic LOS-deficient strain, lpxA, induced equivalent disease severity and similar proinflammatory responses as the serogroup C wild-type parent strain FAM20. This response was abolished in TLR4-/- mice, and neither the wild-type strain of meningococci nor the LOS-deficient mutant was able to cause fatal sepsis in these mice. Mouse survival correlated with low levels of cytokines and chemokines, the chemotactic complement factor C5a and neutrophil levels in blood at 24 h post infection. These data suggest that during meningococcal sepsis the recognition of one or more unidentified non-LOS component(s) by TLR4 is important in stimulating proinflammatory responses, and that fatality associated with meningococcal sepsis in mice is induced by the proinflammatory host response.

Animals↗
PubMed2003

Toll-like receptor expression in human keratinocytes: nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent.

Cultured primary human keratinocytes were screened for their expression of various members of the toll-like receptor (TLR) family. Keratinocytes were found to constitutively express TLR1, TLR2, TLR3, TLR5, and TLR9 but not TLR4, TLR6, TLR7, TLR8, or TLR10 as shown by polymerase chain reaction analysis. The expression of the crucial receptor for signaling of staphylococcal compounds TLR2 was also confirmed by immunohistochemistry, in contrast to TLR4, which showed a negative staining pattern. Next, we analyzed the activation of the proinflammatory nuclear transcription factor kappaB by Staphylococcus aureus strain 8325-4. Using nuclear extract gel shifts, RelA staining, and luciferase reporter transfection plasmids we found a clear induction of nuclear factor kappaB translocation by the bacteria. This translocation induced the transcription of nuclear factor kappaB controlled genes such as inducible nitric oxide synthetase, COX2, and interleukin-8. Transcription of these genes was followed by production of increased amounts of interleukin-8 protein and NO. Inhibition experiments using monoclonal antibodies and the specific platelet activating factor receptor inhibitor CV3988 showed that nuclear factor kappaB activation by S. aureus was TLR2 but not TLR4 or platelet activating factor receptor dependent. In line, the purified staphylococcal cell wall components lipoteichoic acid and peptidoglycan, known to signal through TLR2, also showed nuclear factor kappaB translocation in human keratinocytes, indicating a crucial role of the staphylococcal cell wall in the innate immune stimulation of human keratinocytes. These results help to explain the complex activation of human keratinocytes by S. aureus and its cell wall components in various inflammatory disorders of the skin.

Cells, Cultured↗
PubMed2006

Ethanol suppresses LPS-induced Toll-like receptor 4 clustering, reorganization of the actin cytoskeleton, and associated TNF-alpha production.

BACKGROUND: Ethanol (EtOH) suppresses cytokine responses induced through most Toll-like receptors (TLRs), but the mechanism of action is unclear. We recently found that acute EtOH alters lipopolysaccharide (LPS)-induced partitioning of CD14, a critical component of the LPS receptor complex, within lipid raft fractions in the macrophage-like cell line RAW264.7. METHODS: Here we investigated the role of receptor clustering in alteration of the responses of cells to LPS caused by EtOH both in vitro and in vivo. The cellular distribution of CD14, TLR4, actin cytoskeleton, and tumor necrosis factor-alpha (TNF-alpha) were studied by confocal microscopy following exposure of cells to LPS with or without EtOH. TLR4 and CD14 were clustered into highly colocalized patches on the cell membrane accompanied by the reorganization of the actin cytoskeleton in some of the RAW264.7 cells as well as peritoneal cells following LPS treatment. RESULTS: Addition of EtOH reduced the number of cells that had LPS-induced receptor patches and in which this reorganization occurred. Cells on which CD14 and TLR4 formed clusters or caps had substantially higher levels of membrane-bound TNF-alpha compared with cells without clustering or capping of these molecules. Interference with the actin cytoskeleton by cytochalasin D suppressed the production of TNF-alpha and receptor clustering, as EtOH did. CONCLUSIONS: These data confirm our previous observations, suggest a novel mechanism of EtOH action that involves interference with receptor clustering, and indicate a potential role of actin filaments in the formation of receptor patches, subsequent activation of macrophages by LPS, and production of TNF-alpha.

Actins↗
PubMed2006

Adiponectin inhibits osteoclast formation stimulated by lipopolysaccharide from Actinobacillus actinomycetemcomitans.

Previous epidemiologic studies have suggested that periodontal disease is closely related to obesity and glucose tolerance. As the level of adiponectin, an adipocyte-derived cytokine, in plasma had been reported to decrease in obese and type 2 diabetes patients, we explored the role of adiponectin in the etiology of periodontitis using the D clone of RAW264, a clone that exhibits highly efficient osteoclast formation, to determine whether adiponectin acts as a regulatory molecule in osteoclast formation stimulated by lipopolysaccharide of periodontopathic bacteria. We observed that adiponectin acted as a potent inhibitor of osteoclast formation stimulated by Toll-like receptor 4 (TLR4) ligand and receptor activator of NF-kappaB ligand (RANKL). Because NF-kappaB is an important transcription factor in osteoclast formation, we examined the effect of adiponectin on its transcriptional activity. A luciferase assay showed that adiponectin was able to inhibit the TLR4-mediated NF-kappaB activity in RAW264 cells. In addition, we observed that the cytokine was actually able to inhibit TLR4-mediated expression of the gene for inducible nitric oxide synthase and production of nitric oxide in the cells. These observations strongly suggest that adiponectin may function as a negative regulator of lipopolysaccharide/RANKL-mediated osteoclast formation in periodontal disease.

Actinobacillus Infections↗