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At least 1,189 records · Page 66Linked to original sources
PubMed2005

Regulation of dendritic cell interleukin-12 secretion by tumour cell necrosis.

Dendritic cells (DCs) play a key role in the induction and regulation of antigen-specific immunity. Studies have shown that, similar to infection, cellular necrosis can stimulate DC maturation. However, the ability of necrotic cell death to modulate DC cytokine secretion has yet to be explored. We investigated the regulation of interleukin (IL)-12 secretion by human DCs in response to tumour cell necrosis in an in vitro culture model. Two human tumour cell lines (K562 and JAr) were induced to undergo necrosis using heat injury and repeated cycles of freezing and thawing. Both types of tumour cells tested in this study, when injured, induced secretion of monomeric IL-12p40 by monocyte-derived DCs. Furthermore, priming DCs with necrotic cells augmented IL-12p70 secretion significantly in conjunction with CD40 cross-linking. This was physiologically relevant because cell death-pulsed DCs were more potent than non-pulsed DCs at stimulating T cells to proliferate and secrete interferon (IFN)-gamma. The Toll-like receptor 4 (TLR4) played a role in mediating the DC response to heat-killed, but not freeze/thaw-killed necrotic cells. For both methods of injury, proteins contributed to the effect of necrosis on dendritic cells, whereas DNA was involved in the effect of freeze/thawed cells only. These findings indicate that necrotic tumour cell death is not sufficient to induce bioactive IL-12p70, the Th1 promoting cytokine, but acts to augment its secretion via the CD40/CD40L pathway. The results also highlight that the mode of cell death may determine the mechanism of dendritic cell stimulation.

Animals↗
PubMed2006

Preferential production of the IL-12(p40)/IL-23(p19) heterodimer by dendritic cells from human newborns.

Human newborns present impaired T helper type 1 cell responses, associated with a defect in the synthesis of IL-12 by dendritic cells (DC). IL-23 is a heterodimeric cytokine structurally related to IL-12, implicated in protective and autoimmune responses. We recently showed that upon activation neonatal T cells up-regulate a functional IL-23 receptor and that this cytokine polarizes the differentiation of naive T cells. We therefore investigated the capacity of neonatal DC to secrete IL-23. Lipopolysaccharide (LPS) stimulation induced the transcription of IL-23(p19) mRNA in both adult and neonatal DC, in sharp contrast to the repressed IL-12(p35) gene expression observed in neonatal cells. In comparison to adult DC, neonatal DC produced similar levels of IL-23 protein, in reponse to Toll-like receptor (TLR)-2- and TLR-3 ligands, and higher levels in response to TLR-4- or TLR-8 ligands. The same profile was observed in neonatal mononuclear cells. The supernatant of LPS-stimulated DC induced the secretion of IL-17 by polyclonally activated neonatal CD8(+) T cells, confirming the IL-23 bioactivity. Altogether, these observations strongly suggest that IL-23 could play a role in the immune system of human newborns. In particular, a functional IL-23/IL-17 axis might compensate a suboptimal IL-12/IFN-gamma pathway in early life.

Adult↗
PubMed2006

Toll-like receptor agonists stimulate differential functional activation and cytokine and chemokine gene expression in heterophils isolated from chickens with differential innate responses.

Heterophils isolated from distinct broilers (lines A and B) differ in function and cytokine gene expression profiles. Nothing is known about Toll-like receptor (TLR) expression nor functional activation and cytokine/chemokine gene expression of line A and B heterophils when stimulated with TLR agonists. We found that line A and B heterophils express the same range of TLRs. All the bacterial TLR agonists, peptidoglycan, the synthetic lipoprotein Pam3CSK4, ultra-pure lipopolysaccharide, and flagellin all induced significantly greater functional activation of heterophils from line A compared to B. Only stimulation with the guanosine analog, loxoribine, (LOX) induced a significantly greater functional response in B over A. Additionally, all heterophils from line A stimulated with the bacterial TLR agonists had dramatic upregulation of pro-inflammatory cytokine and chemokine mRNA expression, whereas heterophils from line B had little or no upregulation of these genes. However, stimulation of all heterophils from line B with the bacterial TLR agonists and LOX induced a significant upregulation of IFN-alpha, with little transcription of this cytokine gene in line A heterophils. These findings suggest that the difference in heterophil functional efficiency between these parent lines is due to recognition of pathogens and activation of signaling pathways that induce innate cytokine and chemokine responses.

Animals↗
PubMed2005

Signal transduction in Campylobacter jejuni-induced cytokine production.

Campylobacter jejuni is the leading cause of food-borne illness in the USA and one of the most common causes of diarrhoea worldwide. Central to its pathogenicity is its ability to induce the production of proinflammatory cytokines such as interleukin (IL)-8 in intestinal epithelial cells. Here, we demonstrated that C. jejuni infection of intestinal epithelial cells results in the activation of the ERK and p38 mitogen-activated protein kinases and that the ERK kinase pathway is essential for IL-8 production. We found that MAP kinase stimulation leading to IL-8 secretion requires C. jejuni gene products whose production is stimulated upon contact with epithelial cells. We also found that C. jejuni flagellin is a very poor stimulator of Toll-like receptor (TLR)-5 and therefore does not play a significant role in the stimulation of cytokine production.

Amino Acid Sequence↗
PubMed2002

Gm-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense.

Recent studies in transgenic mice have revealed important insights into the roles of GM-CSF in regulation of surfactant homeostasis and lung host defense. Interruption of the GM-CSF signaling pathway by targeted ablation of the GM-CSF gene or its receptor (GM(-/-) or GM Rbetac(-/-) mice, respectively) resulted in pulmonary alveolar proteinosis (PAP) but no hematologic abnormalities. Alveolar macrophages from GM(-/-) mice have reduced capacity for surfactant catabolism, cell adhesion, phagocytosis, bacterial killing, Toll-receptor signaling, and expression of various pathogen-associated molecular pattern recognition receptors, suggesting arrest at an early stage of differentiation. PAP and abnormalities of alveolar macrophage function were corrected by local expression of GM-CSF in the lung, and expression of the transcription factor PU.1 in alveolar macrophages of GM(-/-) mice rescued most defects. Recently, a strong association of auto-antibodies to GM-CSF or GM-CSF receptor gene mutations with PAP has implicated GM-CSF signaling abnormalities in the pathogenesis of PAP in humans. Together, these observations demonstrate that GM-CSF has a critical role in regulation of surfactant homeostasis and alveolar macrophage innate immune functions in the lung.

Animals↗
PubMed2004

IFN-alpha sensitizes human umbilical vein endothelial cells to apoptosis induced by double-stranded RNA.

The ability of endothelial cells to mount an efficient antiviral response is important in restricting viral dissemination and eliminating viral infection from the endothelium and surrounding tissues. We demonstrate that dsRNA, a molecular signature of viral infection, induced apoptosis in HUVEC, and priming with IFN-alpha shortened the time between when dsRNA was encountered and when apoptosis was initiated. IFN-alpha priming induced higher levels of mRNA for dsRNA-activated protein kinase, 2'5'-oligoadenylate synthetase, and Toll-like receptor 3, transcripts that encode dsRNA-responsive proteins. dsRNA induced activation of dsRNA-activated protein kinase and nuclear translocation of transcription factors RelA and IFN regulatory factor-3 in IFN-alpha-primed HUVECs before the activation of intrinsic and extrinsic apoptotic pathways. These changes did not occur in the absence of dsRNA, and apoptosis resulting from incubation with dsRNA occurred much later when cells were not primed with IFN-alpha. The entire population of IFN-alpha-primed HUVECs underwent nuclear translocation of RelA and IFN regulatory factor-3 in response to dsRNA, whereas less than one-half of the population responded with apoptosis. When IFN-alpha-primed HUVECs were coincubated with dsRNA and proteasome inhibitors, all HUVECs were rendered susceptible to dsRNA-induced apoptosis. These studies provide evidence that many endothelial cells that are alerted to the risk of infection by IFN-alpha would undergo apoptosis sooner in response to dsRNA than non-IFN-alpha-primed cells, and this would enhance the likelihood of eliminating infected cells prior to the production of progeny virions.

Active Transport, Cell Nucleus↗
PubMed2006

Isolation of RNA aptamers against human Toll-like receptor 3 ectodomain.

Toll-like receptor 3 (TLR3) detects double-stranded RNA (dsRNA) known as a universal viral molecular pattern and activates the antiviral immune response. While TLR3 preferentially recognizes polyriboinosine-polyribocytidylic acid (poly (I:C)), a sequence-specific dsRNA has not yet been shown to activate TLR3. To determine whether TLR3 preferentially recognizes some specific sequence that acts on the signaling pathway of TLR3, in vitro selection against human TLR3 ectodomain (TLR3 ECD) was performed. After seventh selection cycle, two major classes, Family-I and -II, were emerged from 64 clones with binding constants of about 3 nM. Although these aptamers bound to TLR3 ECD with high affinity in vitro, they did not have agonist and antagonist effects on TLR3 signaling in TLR3-transfected HEK293 cells. Further analyses of the structure/function relationship of these aptamers will be carried out by mutagenesis, RNase mapping and competition assay using poly (I:C).

Aptamers, Nucleotide↗
PubMed2003

Toll-like receptor-mediated tumor necrosis factor and interleukin-10 production differ during systemic inflammation.

Major trauma is associated with a decreased capacity of patients' leukocytes to produce proinflammatory cytokines on in vitro stimulation. We studied leukocytes from 48 patients with trauma and showed that this hyporeactivity was restricted to gram-negative bacteria, Escherichia coli endotoxin, and unmethylated bacterial DNA, whereas Leptospira interrogans endotoxin-induced tumor necrosis factor production was similar to that observed with healthy donors. As well, tumor necrosis factor and interleukin-6 production in response to gram-positive bacteria was not altered. The expression of toll-like receptor (TLR) 2 was not reduced on patients' monocytes as compared with healthy control subjects, whereas that of TLR4 was reduced. However, the hyporeactivity to gram-negative bacteria and E. coli endotoxin cannot be fully explained by the downregulation of TLR4. Indeed, unlike proinflammatory cytokines, after stimulation with these microbial products the release of antiinflammatory cytokines was increased as compared with healthy control subjects. The increased interleukin-10 production was analyzed in terms of intracellular signaling in peripheral blood mononuclear cells from patients with trauma: our results suggest the involvement of p38 mitogen-activated protein kinase, Sp-1 transcription factor, heterotrimeric Gi protein, and phosphatidylinositol-3'-kinase. In conclusion, the immunodysregulation described for patients with trauma is not a generalized phenomenon but depends on the stimulus and the signaling pathway.

APACHE↗
PubMed2002

MyD88 is involved in the signalling pathway for Taxol-induced apoptosis and TNF-alpha expression in human myelomonocytic cells.

Taxol is an effective anti-tumour drug against a variety of tumour cells. Taxol directly induces apoptosis in addition to a G2/M cell cycle arrest. However, it remains poorly understood how Taxol induces apoptosis in tumour cells. Taxol induces the secretion of inflammatory cytokines in murine macrophages in a toll-like receptor-4 (TLR-4)-dependent manner in addition to its anti-tumour effects, but the effect of Taxol on human macrophages is controversial. In this study, we demonstrated that low doses (less than 1000 nmol/l) of Taxol induced the expression of tumour necrosis factor (TNF)-alpha in human myelomonocytic cells and that the induction of TNF-alpha mRNA was inhibited by dominant-negative myeloid differentiation protein (dnMyD88). Furthermore, we demonstrated that the same doses of Taxol induced apoptosis of the same myelomonocytic cells and that the Taxol-induced apoptosis was also inhibited by dnMyD88. In accordance with the previous reports, Taxol induced the expression of TNF-alpha and apoptosis in a TLR4-independent manner. These results suggest that TNF-alpha expression and apoptosis, both induced by Taxol in human myelomonocytic cells, share the signal transduction molecule MyD88.

Adaptor Proteins, Signal Transducing↗
PubMed2006

The immunomodulator ginsan induces resistance to experimental sepsis by inhibiting Toll-like receptor-mediated inflammatory signals.

Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. In this study, we show that pretreatment of ginsan (25 mug/kg) protected mice from lethality induced by Staphylococcus aureus challenge. This survival benefit was associated with enhanced bacterial clearance from circulation, spleen and kidney. The phagocytic activity of macrophages treated with ginsan was significantly enhanced against S. aureus. However, the production of proinflammatory cytokines, such as TNF-alpha, IL-1beta, IL-6, IFN-gamma, IL-12, and IL-18, was markedly down-regulated in ginsan-treated mice compared with those of control-infected mice. The expression of Toll-like receptor (TLR) 2 and the adaptor molecule MyD88, which was greatly increased in septic macrophages, was significantly reduced by ginsan treatment in vitro. Similarly, the expression of phospho-JNK1/2, phospho-p38 MAPK, and NF-kappaB was decreased in the same culture system. These results illustrate that the antiseptic activity of ginsan can be attributed to enhanced bacterial clearance, and reduced proinflammatory cytokines via the TLR signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Intestinal myofibroblasts in innate immune responses of the intestine.

BACKGROUND & AIMS: Intestinal myofibroblasts are known to respond to inflammatory signals and may play a role in Crohn's disease-associated fibrosis. However, putative involvement by myofibroblasts in innate immune responses as part of intestinal host defense has not been characterized. We therefore analyzed expression and regulation of toll-like receptors (TLRs) in colonic human myofibroblasts (CCD-18) and primary human colonic myofibroblasts in comparison with human lung myofibroblasts (CCD-37). METHODS: Expression of TLRs (1-10) and NOD 1 and 2 was assessed before and after stimulation with either lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by using a custom microarray, reverse-transcription polymerase chain reaction, Northern blot and Western blot analysis, and immunohistochemistry. Activation of signaling pathways, translocation of p65, and secretion of interleukin (IL)-8 were determined. RESULTS: Messenger RNAs encoding for TLR1-9, as well as NOD1 and NOD2, were amplified from cultured and primary human intestinal myofibroblasts. After stimulation with LPS or LTA, a 1.5-4.2-fold up-regulation of TLRs (2, 3, 4, 6, 7) and elements of the signaling cascade (MyD88, TIR domain-containing adapter protein [TIRAP]) was observed. CCD-18 and CCD-37 cells expressed TLR 2 and 4 protein, which were located primarily on the cell membrane. Stimulation with LTA or LPS resulted in activation of the mitogen-activated protein kinases pathway, nuclear translocation of p65, and significantly increased IL-8 secretion. CONCLUSIONS: Bacterial components directly activate intestinal myofibroblasts expressing TLRs. These cells may therefore participate in innate immune responses by sensing and responding to bacterial products that have penetrated into the subepithelial compartment.

Cells, Cultured↗
PubMed2006

TLR signaling pathways: opportunities for activation and blockade in pursuit of therapy.

The identification of the TLRs as key sensors of microbial infection has presented a series of new targets for drug development. The TLRs are linked to the most powerful inflammatory pathways in mammals. The question arises from the start: do we wish to stimulate TLR signaling in order to eradicate specific infections and/or neoplastic diseases? Or do we wish to block TLR signaling to treat inflammatory diseases? If we accept that it would be useful to modulate TLR signaling, the next step is to identify the correct molecular target(s) for the task. Perhaps it might even be possible to exercise selectivity, modulating some aspects of TLR signaling and not others. Classical and reverse genetic analyses offer insight into the possibilities that exist, and point to specific checkpoints within signaling pathways at which modulation might normally be imposed.

Anti-Inflammatory Agents↗
PubMed2005

Stimulation of toll-like receptor 2 with bleomycin results in cellular activation and secretion of pro-inflammatory cytokines and chemokines.

The clinical use of bleomycin results in systemic and pulmonary inflammatory syndromes that are mediated by the production of cytokines and chemokines. In this study, we demonstrate that cell activation is initiated upon the recognition of bleomycin as a pathogen-associated molecular pattern by toll-like receptor (TLR) 2. The THP1 human monocytic cell line, which constitutively expresses high levels of TLR2, secretes interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha during bleomycin exposure. The TLR2-dependent nature of cell activation and cytokine secretion is supported by (1) the inability of TLR2-deficient human embryonic kidney (HEK) 293 cells to exhibit nuclear factor-kappa B (NF-kappaB) activation and secrete IL-8 in response to bleomycin; (2) the acquired ability of HEK293 to exhibit NF-kappaB activation and secrete IL-8 upon experimental expression of TLR2; and (3) the inhibition of cell activation in TLR2-expressing HEK293 and THP1 by anti-TLR2 monoclonal antibody. Collectively, these observations identify TLR2 activation as a critical event that triggers NF-kappaB activation and secretion of cytokines and chemokines during bleomycin exposure. Our in vitro findings could serve as a molecular mechanism underlying the pro-inflammatory toxicity associated with bleomycin. Whether bleomycin engages with other cellular receptors that results in activation of alternate signaling pathways and whether the TLR2-agonist activity of bleomycin contribute to its anti-neoplastic property deserve further study.

Antimetabolites, Antineoplastic↗
PubMed2001

Gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via Toll-like receptors.

Osteoclast differentiation factor (ODF), a recently identified cytokine of the TNF family, is expressed as a membrane-associated protein in osteoblasts and stromal cells. ODF stimulates the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here we investigated the effects of LPS on the gene expression of ODF in mouse osteoblasts and an osteoblast cell line and found that LPS increased the ODF mRNA level. A specific inhibitor of extracellular signal-regulated kinase or protein kinase C inhibited this up-regulation, indicating that extracellular signal-regulated kinase and protein kinase C activation was involved. A protein synthesis inhibitor, cycloheximide, rather enhanced the LPS-mediated increase of ODF mRNA, and both a neutralizing Ab of TNF-alpha and a specific inhibitor of PGE synthesis failed to block the ODF mRNA increase by native LPS. Thus, LPS directly induced ODF mRNA. Mouse osteoblasts and an osteoblast cell line constitutively expressed Toll-like receptor (TLR) 2 and 4, which are known as putative LPS receptors. ODF mRNA increases in response to synthetic lipid A were defective in primary osteoblasts from C3H/HeJ mice that contain a nonfunctional mutation in the TLR4 gene, suggesting that TLR4 plays an essential role in the process. Altogether, our results indicate that ODF gene expression is directly increased in osteoblasts by LPS treatment via TLR, and this pathway may play an important role in the pathogenesis of LPS-mediated bone disorders, such as periodontitis.

Animals↗
PubMed2005

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA.

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.

Active Transport, Cell Nucleus↗
PubMed2006

Neutrophil-derived elastase induces TGF-beta1 secretion in human airway smooth muscle via NF-kappaB pathway.

Neutrophils are infiltrated in airways of individuals with more severe and chronic asthma, with uncertain significance. Airway smooth muscle (ASM), apart from its contractile properties, is critically involved in the pathogenesis of asthma by producing inflammatory mediators. In the present study, we investigated the impact of neutrophil-derived elastase (NE) on ASM in terms of TGF-beta1 release, and we explored the underlying mechanisms. Primary ASM cells were serum starved for 24 h before stimulation with NE (0.01-0.5 microg/ml). TGF-beta1 in supernatant was determined by ELISA and mRNA quantified by real-time RT-QPCR. NF-kappaB nuclear translocation and activation was examined by Western blotting and kappaB-2 dEGFP reporter gene assay. Association of IL-1 receptor-associated kinase (IRAK) with MyD88 was studied by co-immunoprecipitation and Toll-like receptor 4 (TLR4) determined by FACS scan and Western blotting. We demonstrated that NE enhanced TGF-beta1 release in a time-dependent manner. This induction was inhibited by actinomycin D (5 mM), cycloheximide (5 mM), and NF-kappaB inhibitors, including pyrrolidine dithiocarbamate (PDTC, 1 mM), aspirin (2.5 mM), and sodium salyicylate (2.5 mM). Stimulation with NE was rapidly followed by association of IRAK with MyD88, phosphorylation of IkappaBalpha, and nuclear translocation of p65 with increased transactivation activity. We also found that TLR4 levels were reduced upon NE treatment. These data suggest that NE upregulates TGF-beta1 gene expression and release via My88/IRAK/NF-kappaB pathway, possibly through activation of TLR4, and shed light on a potential role of neutrophils in the pathogenesis of asthma.

Cell Line↗
PubMed2004

CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytes.

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. We now show that a novel signal for delaying neutrophil apoptosis is unmethylated CpG motifs prevalent in bacterial DNA (CpG- DNA). Human neutrophils express toll-like receptor 9 that recognizes these motifs. CpG-DNA, but not mammalian DNA or methylated bacterial DNA, markedly enhanced neutrophil viability by delaying spontaneous apoptosis. Endosomal maturation of CpG-DNA is prerequisite for these actions and was coupled to concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to phosphorylation of BAD at Ser112 and Ser136, respectively, and to prevention of decreases in mitochondrial transmembrane potential, cytochrome c release and caspase-3 activation. Consistently, pharmacological inhibition of either ERK or phosphatidylinositol 3-kinase partially reversed these actions of CpG-DNA; however, they did not produce additive inhibition. Furthermore, intravenous injection of CpG-DNA (200 microg/kg) into rats evoked slight decreases in blood pressure and induced a modest leukocytosis, whereas it effectively suppressed neutrophil apoptosis as assayed ex vivo. Our results indicate that unmethylated CpG motifs in bacterial DNA promote neutrophil survival by suppressing the apoptotic machinery and may therefore contribute to prolongation and amplification of inflammation.

Animals↗
PubMed2006

Phosphatidyl inositol-3-phosphate kinase mediates CD14 dependent signaling.

Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is an important mediator of innate immunity and septic shock, but the exact mechanisms mediating cellular LPS recognition and the subsequent translation to inflammatory gene expression remain incompletely understood. CD14 has been established as a receptor that confers high sensitivity to LPS in cells of the myeloid lineage, probably by presenting LPS to Toll receptors. We use an anti CD14 blocking antibody to define a LPS stimulus that activates only this high affinity component of the LPS receptor and then examine CD14 dependent signaling events that are activated in response to LPS stimulation. We describe a novel LPS activated signaling pathway in human PBMC that leads to cytokine production and is mediated by PI3 kinase through Ras and the MEK/ERK cassette. Moreover, we show the PI3 kinase effectors PKB and PKC(zeta) are also activated by PI3 kinase in a CD14 dependent manner in LPS stimulated human PBMC. Thus, PI3 kinase appears to be an essential component in LPS signal transduction.

Antibodies, Monoclonal↗