Endotoxin-induced lung injury in α-galactosylceramide-sensitized mice is caused by failure of interleukin-4 production in lung natural killer T cells.

Administration of bacterial lipopolysaccharide (LPS) known as endotoxin into α-galactosylceramide (α-GalCer)-sensitized mice causes severe lung lesions but few hepatic lesions in lethal shock, and interferon (IFN)-γ is suggested to play a pivotal role in preparation of the lung lesions. In order to clarify the mechanism of how α-GalCer sensitization causes lung lesions exclusively in mice, we examined the differential responsiveness of lungs and livers to α-GalCer sensitization. Although lung and liver natural killer T (NK T) cells both produced IFN-γ in response to α-GalCer, IFN-γ signalling was triggered only in the lungs of α-GalCer-sensitized mice. Lung NK T cells did not produce interleukin (IL)-4 in response to α-GalCer and it did not induce the expression of suppressor of cytokine signalling 1 (SOCS1) in the lungs. Conversely, IL-4 produced by liver NK T cells led to the expression of SOCS1 in the livers of the mice. Neutralization of IL-4 reduced SOCS1 expression in the livers and exacerbated LPS-induced hepatic lesions. IL-10 was produced by liver NK T cells but not lung NK T cells. However, IL-10 was produced constitutively by alveolar epithelial cells in normal lung. Lung NK T cells and liver NK T cells might express CD8 and CD4, respectively. Based on the fact that IL-4 inhibited IFN-γ signalling in the livers of α-GalCer-sensitized mice via SOCS1 expression and signal transducer and activator of transcription 1 (STAT-1) activation, no inhibition of the IFN-γ signalling in the lungs caused LPS-induced lung lesions in α-GalCer-sensitized mice. The detailed mechanism of development of the lung lesions in α-GalCer-sensitized mice is discussed.

Metformin attenuates production of nitric oxide in response to lipopolysaccharide by inhibiting MyD88-independent pathway.

Metformin is reported to ameliorate inflammation in diabetic patients. The effect of metformin on lipopolysaccharide-induced nitric oxide production was studied by using RAW 264.7 macrophage-like cells. The action of metformin was analyzed by dividing lipopolysaccharide signaling into the MyD88-dependent and -independent pathways. Metformin significantly reduced the expression of an inducible type of nitric oxide synthase and inhibited lipopolysaccharide-induced nitric oxide production. On the other hand, metformin did not inhibit lipopolysaccharide-induced tumor necrosis factor-alpha production. The expression levels of interferon-beta protein and mRNA, which is a key molecule in MyD88-independent pathway, were significantly inhibited by metformin. Compound C, a specific AMP-activated protein kinase inhibitor, did not affect the inhibitory action of metformin. Metformin was suggested to inhibit lipopolysaccharide-induced nitric oxide production via inhibition of interferon-beta production in MyD88-independent pathway. Metformin might exhibit an anti- inflammatory action on diabetic complications as well as the antidiabetic action.

Novel mechanism of U18666A-induced tumour necrosis factor-alpha production in RAW 264.7 macrophage cells.

U18666A is a cholesterol transport-inhibiting agent that is used widely to mimic Niemann-Pick type C disease. The effect of U18666A on tumour necrosis factor (TNF)-alpha production in mouse macrophage cell line, RAW 264.7 cells and peritoneal macrophages was examined. U18666A induced TNF-alpha mRNA expression 48 h after the treatment, and TNF-alpha production 48 and 72 h after stimulation in RAW 264.7 cells. U18666A accumulated intracellular free cholesterol in the culture of normal medium but not cholesterol-free medium. U18666A also induced reactive oxygen species (ROS) generation in normal medium but much less in cholesterol-free medium. Anti-oxidant N-acetyl-L-cysteine (NAC) abolished U18666A-induced TNF-alpha production. U18666A led to the phosphorylation of p38 mitogen-activated protein kinase 24 and 48 h after the stimulation and the p38 activation was inhibited in presence of cholesterol-free medium or NAC. A p38 inhibitor reduced U18666A-induced TNF-alpha production. Taken together, U18666A was suggested to induce TNF-alpha production in RAW 264.7 cells via free cholesterol accumulation-mediated ROS generation.

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

The mechanism underlying acute lung injury in lethal endotoxic shock induced by administration of lipopolysaccharide (LPS) into alpha-galactosylceramide (alpha-GalCer)-sensitized mice was studied. Sensitization with alpha-GalCer resulted in the increase of natural killer T (NK T) cells and the production of interferon (IFN)-gamma in the lung. The IFN-gamma 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-gamma antibody inhibited significantly the VCAM-1 expression in alpha-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 alpha-GalCer-sensitized mice. The administration of LPS into alpha-GalCer-sensitized mice caused local production of excessive proinflammatory mediators, such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and nitric oxide. LPS caused microvascular leakage of proteins and cells into bronchoalveolar lavage fluid. Taken together, sensitization with alpha-GalCer was suggested to induce the expression of VCAM-1 via IFN-gamma 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 alpha-GalCer sensitization is discussed.

Lipopolysaccharide augments the in vivo lethal action of doxorubicin against mice via hepatic damage.

The effect of lipopolysaccharide (LPS) on the in vivo lethal action of doxorubicin (DOX) against mice was studied. DOX killed LPS-pretreated mice much earlier than untreated mice, and exhibited a stronger toxic action against LPS-pretreated mice. DOX-induced lethality in LPS-pretreated mice was due to severe hepatic damage, but there were no significant lesions in the heart, kidney and lung. Hepatic lesions were accompanied by caspase 3-positive cells and fragmented DNA-positive cells, suggesting the involvement of apoptosis. DOX induced the production of a high level of interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in LPS-pretreated mice, but not in non-treated mice. The DOX-induced lethality was prevented significantly by anti-IFN-gamma antibody, but not anti-TNF-alpha antibody. Administration of recombinant IFN-gamma in place of LPS augmented definitively the DOX-induced lethality. LPS augmented the DOX-induced lethality in TNF-alpha-deficient mice. Taken together, LPS was suggested to enhance DOX-induced IFN-gamma production and augment the in vivo lethal action via hepatic damage.

Lipopolysaccharide and interferon-gamma enhance Fas-mediated cell death in mouse vascular endothelial cells via augmentation of Fas expression.

The effect of interferon (IFN)-gamma and/or lipopolysaccharide (LPS) on Fas-mediated cell death with anti-Fas agonistic antibody in vascular endothelial cells was examined using a mouse END-D cell line. Anti-Fas agonistic antibody exhibited cytotoxic actions on END-D cells. Fas-mediated cell death was enhanced by LPS or IFN-gamma. The combination of IFN-gamma and LPS significantly enhanced cell death compared to IFN-gamma or LPS alone. IFN-gamma and LPS augmented cell surface expression of Fas, but not tumour necrosis factor (TNF) receptor 1. Inhibitors of p38 mitogen-activated protein kinase (MAPK) prevented augmentation of Fas expression in IFN-gamma and LPS-treated END-D cells. IFN-gamma and LPS-treated END-D cells did not become susceptible to TNF-alpha or nitric oxide-mediated cytotoxicity. IFN-gamma and LPS thus appear to augment selectively Fas expression via activation of p38 MAPK and enhance Fas-mediated cell death in END-D cells. Furthermore, administration of IFN-gamma and LPS into mice induced in vivo expression of Fas on vascular endothelial cells and Fas ligand (FasL) on peripheral blood leucocytes. The relationship between enhancement of Fas-mediated cell death by IFN-gamma and LPS and the development of vascular endothelial injury is discussed.

Mysm1 is required for interferon regulatory factor expression in maintaining HSC quiescence and thymocyte development.

Mysm1(-/-) mice have severely decreased cellularity in hematopoietic organs. We previously revealed that Mysm1 knockout impairs self-renewal and lineage reconstitution of HSCs by abolishing the recruitment of key transcriptional factors to the Gfi-1 locus, an intrinsic regulator of HSC function. The present study further defines a large LSKs in >8-week-old Mysm1(-/-) mice that exhibit increased proliferation and reduced cell lineage differentiation compared with those of WT LSKs. We found that IRF2 and IRF8, which are important for HSC homeostasis and commitment as transcription repressors, were expressed at lower levels in Mysm1(-/-) HSCs, and Mysm1 enhanced function of the IRF2 and IRF8 promoters, suggesting that Mysm1 governs the IRFs for HSC homeostasis. We further found that the lower expressions of IRF2 and IRF8 led to an enhanced transcription of p53 in Mysm1(-/-) HSCs, which was recently defined to have an important role in mediating Mysm1(-/-)-associated defects. The study also revealed that Mysm1(-/-) thymocytes exhibited lower IRF2 expression, but had higher Sca1 expression, which has a role in mediating thymocyte death. Furthermore, we found that the thymocytes from B16 melanoma-bearing mice, which display severe thymus atrophy at late tumor stages, exhibited reduced Mysm1 and IRF2 expression but enhanced Sca1 expression, suggesting that tumors may downregulate Mysm1 and IRF2 for thymic T-cell elimination.