Akiko Morikawa

Augmentation of Nitric Oxide Production by Gamma Interferon in a Mouse Vascular Endothelial Cell Line and Its Modulation by Tumor Necrosis Factor Alpha and Lipopolysaccharide

ABSTRACT The effect of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and lipopolysaccharide (LPS) on nitric oxide (NO) production in the mouse vascular aortic endothelial cell line END-D was examined. LPS, TNF-α, and a low concentration of IFN-γ inhibited NO production in END-D cells, while a high concentration of IFN-γ definitely enhanced it. The NO production induced by a high concentration of IFN-γ was further augmented by using IFN-γ in combination with LPS or TNF-α. In sequential incubations of LPS and IFN-γ, the enhancement of NO production required prior treatment with IFN-γ. Stimulation of END-D cells with a high concentration of IFN-γ led to the expression of inducible NO synthase (iNOS). The augmentation of NO production by IFN-γ alone or in combination with LPS or TNF-α was completely blocked by several inhibitors of iNOS. It was strongly suggested that a high concentration of IFN-γ itself enhanced NO production in END-D cells through inducing the expression of iNOS. LPS and TNF-α exclusively modulated the activity of iNOS once its expression was triggered by IFN-γ. On the other hand, a low concentration of IFN-γ, LPS, and TNF-α reduced NO production through down-regulating constitutive NOS (cNOS). The differential regulation of cNOS- and iNOS-mediated NO production by IFN-γ, TNF-α, and LPS is discussed.

Localization of Apoptosis (Programmed Cell Death) in Mice by Administration of Lipopolysaccharide

Localization of apoptotic cells by administration of lipopolysaccharide into mice was studied by using the in situ specific labeling of fragmented DNA. This method clearly stained the nuclei of thymocytes at the cortex of the thymus. The nuclei of cells in the bone marrow and in the spleen were also positively stained. It was suggested that the cortex in the thymus is where the LPS‐induced programmed cell death occurs.

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.

Augmentation of lipopolysaccharide-induced nitric oxide production by α-galactosylceramide in mouse peritoneal cells

The effect of α-galactosylceramide (α-GalCer) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal cells was studied. α-GalCer augmented LPS-induced NO production in mouse peritoneal cells, but not in RAW 264.7 macrophage cells. α-GalCer augmented NO production, but not tumor necrosis factor (TNF)-α production in LPS-stimulated peritoneal cells. Peritoneal cells produced a significant level of interferon (IFN)-γ in response to α-GalCer and anti-IFN-γ antibody abolished the augmentation of LPS-induced NO production by α-GalCer. Moreover, anti-IFN-γ antibody prevented the enhanced expression of an inducible type of NO synthase mRNA by α-GalCer. α-GalCer did not augment LPS-induced NO production in peritoneal cells from natural killer T (NKT)-deficient mice. Therefore, it was suggested that α-GalCer might augment LPS-induced NO production in peritoneal cells through release of IFN-γ from NKT cells.

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

The effect of interferon (IFN)‐γ 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‐γ. The combination of IFN‐γ and LPS significantly enhanced cell death compared to IFN‐γ or LPS alone. IFN‐γ 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‐γ and LPS‐treated END‐D cells. IFN‐γ and LPS‐treated END‐D cells did not become susceptible to TNF‐α or nitric oxide‐mediated cytotoxicity. IFN‐γ 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‐γ 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‐γ and LPS and the development of vascular endothelial injury 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)‐γ and tumour necrosis factor (TNF)‐α in LPS‐pretreated mice, but not in non‐treated mice. The DOX‐induced lethality was prevented significantly by anti‐IFN‐γ antibody, but not anti‐TNF‐α antibody. Administration of recombinant IFN‐γ in place of LPS augmented definitively the DOX‐induced lethality. LPS augmented the DOX‐induced lethality in TNF‐α‐deficient mice. Taken together, LPS was suggested to enhance DOX‐induced IFN‐γ production and augment the in vivo lethal action via hepatic damage.

Morphological Change in Pseudomonas aeruginosa following Antibiotic Treatment of Experimental Infection in Mice and Its Relation to Susceptibility to Phagocytosis and to Release of Endotoxin

ABSTRACT The relationship between morphological changes in Pseudomonas aeruginosa following antibiotic treatment of experimental infection in mice, susceptibility to phagocytosis, and release of endotoxin was studied. The intraperitoneal administration of P. aeruginosa with imipenem or ceftazidime into mice induced morphological changes in the cells 2 h after injection. Round P. aeruginosa cells with imipenem treatment became susceptible to phagocytosis by peritoneal cells, whereas long filamentous cells with ceftazidime treatment were hardly phagocytized by peritoneal cells. The morphological changes also affected the plasma endotoxin level in the circulation.

Experimental Murine Model for Autoimmune Enterocolitis Using Klebsiella pneumoniae O3 Lipopolysaccharide as a Potent Immunological Adjuvant

An experimental model for autoimmune enterocolitis was produced in mice by repeated immunization of homologous colon extract together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes in the intestinal lesions were characterized by infiltration with polymorphonuclear leukocytes in the lamina propria, muscularis mucosae and submucosa of repeatedly immunized mice. No such intestinal lesions were produced in mice receiving injections of colon extract alone or KO3 LPS alone. Development of the autoantibody and delayed‐type hypersensitivity against colon extract were found in mice immunized with the mixture of colon extract and KO3 LPS. Distinct positive staining was detected specifically on the columnar epithelium of villi. Sera from hyperimmunized mice defined organ‐specific antigens present in the intestine. Therefore, it was suggested that the intestinal lesions might be caused by an autoimmune mechanism.

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide‐induced nitric oxide production was not inhibited, but rather increased in the presence of a non‐cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin‐induced NO production seems to occur via the iκB/NF‐κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na‐K pump, we examined whether the Na‐K pump inhibitor amiloride suppresses nystatin‐induced NO production. It was found that amiloride significantly inhibited nystatin‐induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na‐pump activation through the PI3 kinase/Akt/NF‐κB pathway without affecting the condition of lipid rafts.

Differences in the mechanism of nitric oxide production between mouse vascular endothelial cells and macrophages.

The detailed mechanism of NO production in mouse vascular endothelial cells, END-D, was studied. The NO production in END-D cells was triggered by gamma interferon (IFN-γ), but not LPS. However, LPS augmented the NO production in IFN-γ-stimulated END-D cells. A high level of NO production was due to the expression of an inducible type of NO synthase (iNOS) in those cells. A significant amount of NO was detected 18 h after IFN-γ stimulation, accompanied by the delayed iNOS expression. The JAK/STAT signal pathway mediated IFN-γ-induced NO production, but did not participate in the LPS-induced augmentation. Further, no activation of nuclear factor (NF)-κB was involved in the NO production in END-D cells stimulated with either IFN-γ and/or LPS. The mechanism of NO production in END-D cells was suggested to be different from that in mouse macrophages. The differential regulation of NO production in mouse vascular endothelial cells and macrophages is discussed.