Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha
Fatema B. AlmughlliqYong Qin KohHassendrini N. PeirisKanchan VaswaniBuddhika J. ArachchigeSarah ReedMurray D. Mitchell
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Abstract A cell-by-cell analysis of the secretory ability of stimulated, individual alveolar macrophages (AMs) was performed through use of a tumor necrosis factor a (TNF-α) reverse hemolytic plaque assay. Two functional end points were measured: the percentage of AMs that were TNF-α secretors and the cumulative amount of TNF-α secreted by AMs (average plaque area, μm2). Lipopolysaccharide (LPS; 100 μg/ml) increased cumulative TNF-α release at both 7 and 20 h of incubation. On the other hand, a phorbol ester (phorbol myristate acetate, PMA) stimulated TNF-α release at 20 h of incubation but not at 7 h. Under nonstimulated culture conditions, 5-10% of all AMs released detectable TNF-α. PMA (but not LPS) induced a significant increase in the fraction of AMs capable of releasing TNF-α (15.1 ± 1.1% vs. 9.0 ± 1.6%, PMA vs. control, P < .05). Differences in the time course of secreted TNF-α, together with the recruiting effect of PMA, suggest that LPS and PMA target TNF-α- secretory subpopulations of AMs that differ in number and secretory characteristics.
Pulmonary alveolus
Phorbol
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These experiments provide an explanation for the observation that two intravenous injections of lipopolysaccharide (LPS) spaced 5 h apart in rabbits cause tumor necrosis factor/cachectin (TNF) levels to rise in the blood only after the first LPS injection. Herein we show that treatment of elicited peritoneal exudate rabbit macrophages (PEM) with two doses of LPS given 9 h apart results in a marked reduction in TNF production by the second LPS exposure. This state of hyporesponsiveness is a result of adaptation to LPS, is induced by LPS concentrations that are 1,000-fold less than required to induce TNF production (picograms vs. nanograms), is characterized by a decrease in LPS-induced TNF mRNA without any change in TNF mRNA half-life, is not changed by including indomethacin in cultures, and is specific for LPS since LPS-adapted cells display a TNF response to heat-killed Staphylococcus aureus that is at least as good as that observed in control PEM.
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E5531, a synthetic lipid A analog, has been shown to inhibit endotoxin (lipopolysaccharide, LPS)-induced tumor necrosis factor-α (TNF-α) production by human monocytes and murine macrophages. Whether it also inhibits LPS induction of manganese superoxide dismutase (MnSOD) is not clear. In the current study, we demonstrated that E5531, while having no effect on TNF-α and MnSOD mRNAs by itself, markedly inhibited LPS- and lipid A-, but not TNF-α-, induced increases in TNF-α and MnSOD mRNAs in human monocytes. In contrast, E5531 at concentrations and conditions that markedly inhibit LPS-induced increases in TNF-α and MnSOD mRNAs, and TNFα production by human monocytes, had no effect on murine peritoneal macrophages. These results demonstrate that E5531 is a potent LPS antagonist in human monocytes. However, it does not show antagonist action against LPS in murine macrophages in the range of concentrations tested, suggesting that E5531 is a more potent antagonist in humans than in mice.
Lipid A
Monocyte
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Lethality and tumor necrosis factor production induced by different types of lipopolysaccharide were studied in naive (non-primed) rats during the late phase of endotoxin tolerance. The correlation with antilipopolysaccharide antibodies was also analyzed. No correlation was found between tumor necrosis factor levels and lipopolysaccharide-induced mortality in naive animals. Low-toxicity lipopolysaccharide preparations induced levels of tumor necrosis factor similar to those induced with more toxic types of lipopolysaccharide. Late tolerance was associated with progressively lower levels of lipopolysaccharide-induced tumor necrosis factor and increasing titers of antilipopolysaccharide antibodies after repeated injections of homologous lipopolysaccharide. During late endotonxin tolerance, a direct correlation between the lipopolysaccharide dose and peak tumor necrosis factor serum levels was found. We conclude that since tumor necrosis factor serum levels do not correlate with mortality, tumor necrosis factor alone cannot explain the lethal effect of lipopolysaccharide.
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Alveolar macrophage
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Optimal activation of human monocytes in vitro for the biosynthesis of tumor necrosis factor was achieved only with complete S-form lipopolysaccharide. Endotoxin preparations with shorter carbohydrate chains or the lipid A component of lipopolysaccharide were not able to induce release of comparable amounts of tumor necrosis factor by monocytes under the conditions described. The same differences in the level of tumor necrosis factor mRNA were observed. Moreover, addition of these agents to appropriate monocyte-activating substances inhibited the production of tumor necrosis factor. The regulatory implications of this phenomenon are discussed.
Monocyte
Lipid A
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Macrophages are induced by LPS to release a number of products that determine the host response during gram negative sepsis. To examine the role of one such substance, tumor necrosis factor (TNF), in mediating LPS-induced injury, we employed a rabbit model of endotoxic shock to (a) determine the kinetics and extent of release of TNF into plasma after injection of LPS, and (b) to evaluate the protective effect of in vivo neutralization of LPS-induced TNF by prior infusion of anti-TNF antibody. TNF was maximally induced 45-100 min after injection of 10 micrograms i.v. parent Salmonella minnesota Re595 LPS or 250 micrograms Re595 LPS-HDL complexes. Maximal induction of TNF by LPS was associated with development of hypotension, focal hepatic necrosis, intravascular fibrin deposition and lethality. Based on (a) the peak levels of TNF observed in serum, 2.5 X 10(3) U/ml, (b) the specific activity of purified rabbit macrophage-derived TNF, 1 X 10(8) U/mg, and (c) the biphasic disappearance of intravenously injected purified TNF (t1/2 = 0.5 min, 11 min) we constructed a kinetic model showing that at least 130 micrograms of TNF (1.3 X 10(7) U) was released into plasma 30-200 min postinjection of LPS. Prior infusion of anti-TNF antibody (30-45 min before LPS injection) resulted in neutralization of the LPS-induced serum TNF activity and provided significant protection from the development of hypotension, fibrin deposition, and lethality. Thus, these results provide further evidence that TNF plays a central role mediating the pathophysiologic changes that occur during gram negative endotoxic shock.
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This work demonstrates the need for the continued presence of lipopolysaccharide (LPS) for tumor necrosis factor (TNF) production by thioglycollate-induced peritoneal macrophages. Removal of LPS at any time resulted in the abrupt cessation of further TNF production. The readdition of LPS resulted in further production of TNF but the yield was limited to the amount that would have been produced had the LPS not been removed.
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Lipid A
Strain (injury)
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AIM To investigate activation of nuclear factor κB (NF κB) and the expression of tumor necrosis factor α (TNF α) in lipopolysaccharide induced pulmonary alveolar Macrophages (PAM) stimulated with Nitric Oxide. METHODS PAM collected by BALT were cultured and divided into three groups: Control group, LPS stimulated group, and NO+LPS group. The NF κB activity of nuclear protein extract from the PAM and the concentration of TNF α in the supernatant were measured by electrophoretic mobility shift assay and ELISA. RESULTS The activity of NF κB and level of TNF α significantly increased 24 h after LPS stimulation; Compared with LPS stimulated group, both NF κB activity and concentration of TNF α were significantly lowered in NO+LPS group. CONCLUSION LPS might activate NF κB in the PAMs and induce the increase of transcription and expression of TNF α gene; NO could inhibit the activation of NF κB and reduce the release of TNF α.
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