Shamima Islam

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.

Lethal endotoxic shock using α -galactosylceramide sensitization as a new experimental model of septic shock

The effect of α-galactosylceramide (α-GalCer) on lipopolysaccharide (LPS)-mediated lethality was examined. Administration of LPS killed all mice pretreated with α-GalCer, but not untreated control mice. The lethal shock in α-GalCer-sensitized mice was accompanied by severe pulmonary lesions with marked infiltration of inflammatory cells and massive cell death. On the other hand, hepatic lesions were focal and mild. A number of cells in pulmonary and hepatic lesions underwent apoptotic cell death. α-GalCer sensitization was ineffective for the development of the systemic lethal shock in Vα14-positive natural killer T cell-deficient mice. Sensitization with α-GalCer led to the circulation of a high level of interferon (IFN)-γ and further augmented the production of tumor necrosis factor (TNF)-α in response to LPS. The lethal shock was abolished by the administration of anti-IFN-γ or TNF-α antibody. Further, the lethal shock did not occur in TNF-α-deficient mice. Taken together, α-GalCer sensitization rendered mice very susceptible to LPS-mediated lethal shock, and IFN-γ and TNF-α were found to play a critical role in the preparation and execution of the systemic lethal shock, respectively. The LPS-mediated lethal shock using α-GalCer sensitization might be useful for researchers employing experimental models of sepsis and septic shock.

Piceatannol Prevents Lipopolysaccharide (LPS)-Induced Nitric Oxide (NO) Production and Nuclear Factor (NF)-κB Activation by Inhibiting IκB Kinase (IKK)

The effect of piceatannol on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS‐stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down‐regulation of LPS‐induced nuclear factor (NF)‐κB activation, but not by its cytotoxic action. Piceatannol inhibited IκB kinase (IKK)‐α and β phosphorylation, and subsequently IKB‐α phosphorylation in LPS‐stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen‐activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress‐activated protein kinase/c‐Jun NH2‐terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf‐1 molecules, which regulated the activation of IKK‐α and β phosphorylation. The detailed mechanism of the inhibition of LPS‐induced NO production by piceatannol is discussed.

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.

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.

Lipopolysaccharide Prevents Doxorubicin-Induced Apoptosis in RAW 264.7 Macrophage Cells by Inhibiting p53 Activation

The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.

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.

Characterization of Biological Activities of Brucella melitensis Lipopolysaccharide

Biological activities of lipopolysaccharide (LPS) from Brucella melitensis 16M were characterized in comparison with LPS from Escherichia coli O55. LPS extracted from B. melitensis was smooth type by electrophoretic analysis with silver staining. The endotoxin‐specific Limulus activity of B. melitensis LPS was lower than that of E. coli LPS. There was no significant production of tumor necrosis factor‐α and nitric oxide in RAW 264.7 macrophage cells stimulated with B. melitensis LPS, although E. coli LPS definitely induced their production. On the other hand, B. melitensis LPS exhibited a higher anti‐complement activity than E. coli LPS. B. melitensis LPS as well as E. coli LPS exhibited a strong adjuvant action on antibody response to bovine serum. The characteristic biological activities of B. melitensis are 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.

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.