Yoshikazu Naiki

Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide

BackgroundRecently it has been reported that, toll-like receptors (TLRs) are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS) via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line.MethodsExpression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis, respectively. Activation of nuclear factor (NF)-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP) kinase and interferon regulatory factor (IRF)-3 was detected by immunoblot analysis.ResultsHuman NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS.ConclusionAlthough human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3.

Thalidomide inhibits lipopolysaccharide-induced tumor necrosis factor-α production via down-regulation of MyD88 expression

The effect of thalidomide on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production was studied by using RAW 264.7 murine macrophage-like cells. Thalidomide significantly inhibited LPS-induced TNF-α production. Thalidomide prevented the activation of nuclear factor (NF)-KB by down-regulating phosphorylation of inhibitory KB factor (IKB), and IKB kinase (IKK)-α and IKK-β Moreover, thalidomide inhibited LPS-induced phosphorylation of AKT, p38 and stress-activated protein kinase (SAPK)/JNK. The expression of myeloid differentiation factor 88 (MyD88) protein and mRNA was markedly reduced in thalidomide-treated RAW 264.7 cells but there was no significant alteration in the expression of interleukin-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6 in the cells. Thalidomide did not affect the cell surface expression of Toll-like receptor (TLR) 4 and CD14, suggesting the impairment of intracellular LPS signalling in thalidomide-treated RAW 264.7 cells. Thalidomide significantly inhibited the TNF-α production in response to palmitoyl-Cys(RS)-2,3-di(palmitoyloxy) propyl)-Ala-Gly-OH (Pam3Cys) as a MyD88-dependent TLR2 ligand. Therefore, it is suggested that thalidomide might impair LPS signalling via down-regulation of MyD88 protein and mRNA and inhibit LPS-induced TNF-α production. The putative mechanism of thalidomide-induced MyD88 down-regulation is discussed.

Astrocyte elevated gene‐1 (AEG‐1) is induced by lipopolysaccharide as toll‐like receptor 4 (TLR4) ligand and regulates TLR4 signalling

Astrocyte elevated gene‐1 (AEG‐1) is induced by human immunodeficiency virus 1 (HIV‐1) infection and involved in tumour progression, migration and invasion as a nuclear factor‐κB (NF‐κB) ‐dependent gene. The involvement of AEG‐1 on lipopolysaccharide (LPS) ‐induced proinflammatory cytokine production was examined. AEG‐1 was induced via NF‐κB activation in LPS‐stimulated U937 human promonocytic cells. AEG‐1 induced by LPS subsequently regulated NF‐κB activation. The prevention of AEG‐1 expression inhibited LPS‐induced tumour necrosis factor‐α and prostaglandin E2 production. The AEG‐1 activation was not induced by toll‐like receptor ligands other than LPS. Therefore, AEG‐1 was suggested to be a LPS‐responsive gene and involved in LPS‐induced inflammatory response.

The mechanism of development of acute lung injury in lethal endotoxic shock using α-galactosylceramide sensitization

The mechanism underlying acute lung injury in lethal endotoxic shock induced by administration of lipopolysaccharide (LPS) into α‐galactosylceramide (α‐GalCer)‐sensitized mice was studied. Sensitization with α‐GalCer resulted in the increase of natural killer T (NK T) cells and the production of interferon (IFN)‐γ in the lung. The IFN‐γ that was produced induced expression of adhesion molecules, especially vascular cell adhesion molecule‐1 (VCAM‐1), on vascular endothelial cells in the lung. Anti‐IFN‐γ antibody inhibited significantly the VCAM‐1 expression in α‐GalCer‐sensitized mice. Very late activating antigen‐4‐positive cells, as the counterpart of VCAM‐1, accumulated in the lung. Anti‐VCAM‐1 antibody prevented LPS‐mediated lethal shock in α‐GalCer‐sensitized mice. The administration of LPS into α‐GalCer‐sensitized mice caused local production of excessive proinflammatory mediators, such as tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 and nitric oxide. LPS caused microvascular leakage of proteins and cells into bronchoalveolar lavage fluid. Taken together, sensitization with α‐GalCer was suggested to induce the expression of VCAM‐1 via IFN‐γ produced by NK T cells and recruit a number of inflammatory cells into the lung. Further, LPS was suggested to lead to the production of excessive proinflammatory mediators, the elevation of pulmonary permeability and cell death. The putative mechanism of acute lung injury in LPS‐mediated lethal shock using α‐GalCer sensitization is discussed.

Interleukin-10 inhibits tumor necrosis factor-α production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-α production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-α production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-κB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-α production via reduced MyD88 expression.

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5(+) IgM(+) WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-kappaB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.

Receptor activator of nuclear factor-kappa B ligand induces osteoclast formation in RAW 264.7 macrophage cells via augmented production of macrophage–colony-stimulating factor

RAW 264.7 macrophage cells differentiate into osteoclast‐like cells in the presence of RANKL. Participation of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells was examined. TRAP‐positive osteoclast‐like cells appeared in RAW 264.7 cells cultured in the presence of RANKL. RANKL‐induced osteoclast formation was markedly inhibited by anti‐M‐CSF antibody. RANKL augmented M‐CSF mRNA expression and M‐CSF production in RAW 264.7 cells. Further, anti‐M‐CSF antibody inhibited the expression of RANK, c‐fms, c‐fos and TRAP mRNA in RANKL‐stimulated RAW 264.7 cells. However, anti‐M‐CSF antibody did not affect the expression of DC‐STAMP in the stimulated cells. Therefore, RANKL was suggested to induce osteoclast formation in RAW 264.7 cells via augmented production of M‐CSF. The putative role of M‐CSF in RANKL‐induced osteoclast formation of RAW 264.7 cells is discussed.

Interleukin (IL)-10 attenuates lipopolysaccharide-induced IL-6 production via inhibition of IκB-ζ activity by Bcl-3

The inhibitory effect of interleukin-10 (IL-10), an anti-inflammatory cytokine, on lipopolysaccharide (LPS)-induced IL-6 production was characterized by simultaneous stimulation of RAW 264.7 cells with LPS and IL-10. The presence of IL-10 significantly inhibited LPS-induced IL-6 production at a transcriptional level. The expression of IκB-ζ, which promotes IL-6 production, was induced in response to LPS and it was definitely suppressed in the presence of IL-10. Further, IL-10 inhibited LPS-induced NF-κB activation. A pharmacological inhibitor of NF-κB prevented LPS-induced IκB-ζ expression, suggesting that IL-10 might inhibit LPS-induced IκB-ζ expression via the inactivation of NF-κB. In LPS- and IL-10-stimulated cells, the expression of Bcl-3 that inhibits NF-κB activation was significantly augmented. Introduction of Bcl-3 siRNA abolished IL-10-mediated IκB-ζ inhibition. In the presence of Bcl-3, siRNA IL-10 failed to inhibit LPS-induced IL-6 production. Therefore, it was suggested that Bcl-3 induced by IL-10 might reduce LPS-induced IκB-ζ activity via inactivation of NF-κB and that reduced IκB-ζ activity failed to promote LPS-induced IL-6 production.

Lipopolysaccharide enhances interferon-γ-induced nitric oxide (NO) production in murine vascular endothelial cells via augmentation of interferon regulatory factor-1 activation

Lipopolysaccharide (LPS) enhances the production of nitric oxide (NO) in interferon (IFN)-γstimulated vascular endothelial cells. We studied the mechanism by which LPS enhances IFN-γ-induced NO production by using the murine vascular endothelial cell line, END-D. LPS enhanced IFN-γinduced NO production via augmented expression of inducible type NO synthase (iNOS) mRNA. LPS significantly augmented the activation of interferon regulatory factor (IRF)-1 in IFN-γ-stimulated END-D cells, although it did not affect the activation of either MyD88-dependent nuclear factor (NF)-κB or MyD88-independent IRF-3. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), prevented the nuclear translocation of IRF-1 in LPS and IFN-γ-stimulated END-D cells, and inhibited the iNOS expression and NO production in those cells. Therefore, it is proposed that LPS enhanced NO production in IFN-γ-stimulated END-D cells via augmenting p38 MAPKmediated IRF-1 activation.

Involvement of interleukin-1 receptor-associated kinase (IRAK)-M in toll-like receptor (TLR) 7-mediated tolerance in RAW 264.7 macrophage-like cells

The effect of toll-like receptor (TLR) 7 ligand pretreatment on the production of tumor necrosis factor (TNF)-alpha in response to TLR7 or TLR2 ligand was examined in order to establish a new TLR-mediated tolerance. RAW 264.7 macrophage-like cells were treated with imiquimod R837 as a TLR7 ligand for 18h, washed and incubated in fresh culture medium 6h. The second challenge with imiquimod R837 as a TLR7 ligand or Pam3CysSK4 as a TLR2 ligand resulted in reduced TNF-alpha production in TLR7 ligand-pretreated cells. There was impaired activation of NF-kappaB, p38 and stress-activated protein kinase (SAPK) in the tolerant cells. The expression of IRAK-M as a negative regulator of TLR signaling was markedly augmented in the tolerant cells while the interleukin-1 receptor-associated kinase (IRAK)-1 functioned normally. The involvement of IRAK-M in the TLR7-mediated tolerance is discussed.