Kazuko Takahashi

The inhibitory action of quercetin on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of quercetin on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied. Quercetin pretreatment significantly inhibited NO production in an LPS-stimulated RAW 264.7 murine macrophage cell line. Post-treatment with quercetin partially inhibited NO production. The inhibitory action of quercetin was due to neither the cytotoxic action nor altered LPS binding. The expression of inducible-type NO synthase (iNOS) was markedly down-regulated by quercetin. Quercetin suppressed the release of free nuclear factor (NF)-κB by preventing degradation of IκB-α and IκB-β. Moreover, quercetin blocked the phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2), p38, and c-Jun NH 2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and, further, the activity of tyrosine kinases in LPS-stimulated RAW cells. Quercetin also inhibited interferon (IFN)-γ-induced NO production. Taken together, these results indicate that the inhibitory action of quercetin on NO production in LPS- and/or IFN-γ-stimulated macrophages might be due to abrogation of iNOS protein induction by impairment of a series of intracellular signal pathways.

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.

Protective Effect of Wogonin on Endotoxin‐Induced Lethal Shock in D‐Galactosamine‐Sensitized Mice

The effects of wogonin, a major flavonoid from Scutellaria baicalensis Georgi, on lipopolysaccharide (LPS)‐induced lethal shock in mice was investigated. Wogonin pretreatment prevented the lethal shock in mice injected with D‐galactosamine (D‐GalN) and LPS, but not in mice injected with a high dose of LPS. Wogonin definitely inhibited the hepatic injury in mice injected with D‐GalN, and LPS and reduced the level of circulating tumor necrosis factor (TNF)‐α. The reduction was more marked in mice injected with D‐GalN and LPS compared with that in mice injected with a high dose of LPS. Wogonin pretreatment did not inhibit the lipid peroxidation in mice receiving either D‐GalN and LPS or a high dose of LPS. Wogonin inhibited the in vitro production of TNF‐α and nitric oxide in LPS‐stimulated RAW 264.7 cells. The mechanism of the protective effect of wogonin on the lethal shock in mice injected with D‐GalN and LPS is discussed.

Detection of lipopolysaccharide (LPS) and identification of its serotype by an enzyme-linked immunosorbent assay (ELISA) using poly-l-lysine

A new solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of LPS and identification of its serotype with antisera. Since LPS binds poorly to polystyrene microplates, precoating with poly-L-lysine was used before coating LPS on the surface of microplates. The small amount of LPS in complex mixtures (i.e., less than 1 microgram/ml) could be detectable in ELISA. Use of poly-L-lysine with high molecular weight (MW) provided a higher sensitivity than poly-L-lysine with low MW. Precoating with polymyxin B, or poly-L-histidine was less effective in the sensitivity than precoating with poly-L-lysine, but it was still better than no precoating. The newly developed ELISA technique could be also applied for detection of anti-LPS antibodies in sera or for screening of monoclonal anti-LPS antibody.

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.

Production of murine collagen-induced arthritis using Klebsiella pneumoniae O3 lipopolysaccharide as a potent immunological adjuvant

Collagen‐induced arthritis (CIA) was produced in mice with non H‐2q and H‐2r haplotypes by repeated immunization of porcine type‐II collagen (CII) together with Klebsiella O3 lipopolysaccharide (KO3 LPS) as an immunological adjuvant. Histological changes that appeared in joints of repeatedly immunized mice were characterized by destruction of normal joint structure, synovial hyperplasia with proliferation of synovial cells, and infiltration of inflammatory cells. No such lesions were produced in mice receiving repeated injections of CII alone or KO3 LPS alone. Development of the humoral antibody and the delayed‐type hypersensitivity to CII was exclusively found in mice immunized with the mixture of CII and KO3 LPS. It was therefore suggested that arthritis lesions induced by repeated immunization with the mixture of CII and KO3 LPS might be caused by an autoimmune mechanism, and that the experimental model might be useful for characterization of human rheumatoid arthritis (RA).

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.

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.

Increased Phosphodiesters in Lipopolysaccharide Prepared from the Polymyxin B-Resistant Isolate of Klebsiella pneumoniae

A polymyxin B (PXB)‐resistant mutant of Klebsiella pneumoniae O3 was isolated. Lipopolysaccharide (LPS) extracted from the PXB‐resistant isolate bound little PXB, although LPS from the parental strain did. The 31P nuclear magnetic resonance (NMR) spectrum of PXB‐resistant type LPS showed that it contained much less of the phosphomonoesters and the pyrophosphate esters, and an increased amount of the phosphodiesters, compared to the parental type LPS. The decrease in the binding of PXB might be due to altered phosphate groups on the PXB‐resistant type LPS, suggesting that it might explain the PXB‐resistance of the mutant.

GSK2656157, a PERK inhibitor, reduced LPS-induced IL-1β production through inhibiting Caspase 1 activation in macrophage-like J774.1 cells

Abstract IL-1β is one of the inflammatory cytokines and is cleaved from pro-IL-1β proteolytically by activated Caspase 1. For the activation of Caspase 1, inflammasome was formed by two signals, what is called, priming and triggering signals. In this study, it was found that mouse macrophage J774.1 cells, when treated by single large amount of lipopolysaccharide (LPS), produced a significant amount of IL-1β. On the other hand, IL-1β production was not detected when treated by a single, small amount of LPS. Then, focusing on endoplasmic reticulum (ER) stress response among stress responses induced by a large amount of LPS, when GSK2656157, a PERK inhibitor, was used for inhibition of ER stress, GSK2656157 reduced IL-1β production dose-dependently. Next, when Thapsigargin, an ER stress reagent, was added with LPS, IL-1β production increased more than by LPS alone. Thus, these results suggested that ER stress was involved in LPS-induced IL-1β production. When the activation of Caspase 1 was examined by fluorescence activated cell sorter analysis, it was found that GSK2656157 inhibited LPS-induced Caspase 1 activation. Further, it was confirmed that GSK2656157 did not affect LPS-induced TNF-α production and activation of NF-κB and specifically inhibited the PERK/eIF-2α pathway. Therefore, it was found that GSK2656157 specifically inhibited ER stress induced by large amount of LPS and reduced LPS-induced IL-1β production through inhibition of Caspase 1 activation.