Ken-ichi Tanamoto

Salmonella-Type Heptaacylated Lipid A Is Inactive and Acts as an Antagonist of Lipopolysaccharide Action on Human Line Cells

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-α induction and NF-κB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-α release and activated NF-κB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-α and NF-κB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-α induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-α release and NF-κB activation in THP-1 cells.

Endotoxic Properties of Free Lipid a from Porphyromonas Gingivalis

The relationship between chemical structure and biological activity of the lipid A from Porphyromonas gingivalis, which we recently isolated and whose complete chemical structure was determined [Kumada et al. (1995). J Bacteriol 177, 2098-2106], was studied. The lipid A exhibited endotoxic activity in all the assay systems tested: Limulus gelation activity, lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells and induction of nitric oxide (NO) and tumour necrosis factor alpha (TNF) release from both mouse peritoneal macrophages and the J774-1 mouse macrophage-like cell line. The activity was, however, about 100-fold less than that of Salmonella minnesota LPS used as a control. The moderate activity of the lipid A may be partially explained by its unique fatty acid composition and the lack of a phosphate group in position 4. In contrast, the lipid A as well as whole LPS of P. gingivalis unexpectedly exhibited an even stronger induction of TNF from the human monocytic THP-1 cell line than control LPS when measured by the minimum stimulatory dose. The difference in sensitivity of human and mouse cells to P. gingivalis lipid A suggests that the recognition mechanism, including that for the receptor for endotoxin, may be regulated in different ways in the two cells.

Predominant role of the substituents on the hydroxyl groups of 3‐hydroxy fatty acids of non‐reducing glucosamine in lipid A for the endotoxic and antagonistic activity

The synthetic disaccharide precursor of lipid A (406: identical to lipid IVA) was found to reduce its endotoxic activity in mice by an order of 105 or more, by replacing the hydroxyl groups with succinyl or acetyl residues. Both the succinylated and acetylated 406 were also found to antagonize the endotoxic mitogenicity on murine splenocytes. Previous studies demonstrated that the succinylated or acetylated synthetic complete lipid A preparations retained the whole endotoxic activity [1994, Infect. Immunol. 62, 1705]. The drastic contrast in all of these results suggests the importance of the substituents on the hydroxyl groups of 3‐hydroxy fatty acids of non‐reducing glucosamine of lipid A for the activity and for transformation to the antagonistic structure.

Succinylated lipid A is a potent and specific inhibitor of endotoxin mitogenicity

Chemically modified lipopolysaccharides of Salmonella abortus-equi were tested for mitogenicity on mouse spleen cells as well as antagonism of the mitogenicity of intact lipopolysaccharide (LPS). All the lipopolysaccharide preparations deacylated by different alkaline treatments suffered a drastic loss of mitogenicity. The mitogenic activity of lipid A was also lost when succinic residues were introduced on hydroxyl groups. Partially deacylated alkaline-treated preparations (but not completely deacylated preparations) inhibited the activation of splenic B-cells by LPS. They were found to be toxic to spleen cells, however, and to suppress not only the mitogenicity of LPS but that of concanavalin A as well. This inhibitory action was not exhibited when all of the fatty acid was eliminated. Succinylated lipid A, on the other hand, was not toxic to the cells and inhibited the B-cell mitogenicity of lipopolysaccharide (but not the T-cell mitogenicity of concanavalin A). Chemical analysis revealed that about 4.6 mol of succinic acid had been introduced into lipid A by succinylation, and that the fatty acid and phosphate composition was unchanged by this treatment. Macrophages do not seem to participate in this inhibition. Inhibition was observed when succinylated lipid A was added either at the same time or after lipid A mitogen, but optimal inhibition was expressed when it was added to the culture 3 h before LPS. Inhibition was not affected by washing the cells before adding LPS. Inhibition increased as the ratio of suppressor to mitogen increased, suggesting that the succinylated lipid A competes with intact LPS.

Induction of prostaglandin release from macrophages by bacterial endotoxin

Publisher Summary This chapter discusses the induction of prostaglandin release from macrophages by bacterial endotoxin. Endotoxin is chemically a lipopolysaccharide (LPS), consisting of a hydrophilic polysaccharide part and a hydrophobic lipid part, named lipid A, which has been proven to be the active center of almost all the biological activities of endotoxin. The concept of involvement of prostaglandins in endotoxic action is supported partly by the observation that certain endotoxic activities such as the induction of fever, abortion, and the early phase of endotoxic shock are suppressed by an inhibitor of cyclooxygenase. It has also been suggested that endotoxin shock is closely related to increased concentrations of several groups of prostanoids in blood and lymph. The chapter describes the preparation of bacterial endotoxin, cell (macrophage) preparation, and measurement of prostaglandins. Prostaglandins and their metabolites are generally measured by such techniques as radioimmunoassay (RIA), gas chromatography (GC), GC–mass spectrometry, and high-performance liquid chromatography (HPLC).

Anti-endotoxin properties of a cinnamon bark-derived compound and its effect on the endotoxin shock model

An endotoxin inhibitor derived from cinnamon bark was characterized chemically and tested for anti-LPS properties. Chemical analysis suggested that the active center of the inhibitor was in the lipid portion. Upon incubation with LPS molecule, the inhibitor reduced the ability of LPS to induce TNFα and generate nitric oxide from various cells in vitro, and Limulus gelation activity. The lethal toxicity of LPS in galactosamine-sensitized mice and pyrogenicity of LPS in a rabbit model were reduced 1000- and 100-fold by pre-incubation with the inhibitor, respectively. Simultaneous but separate injection of the inhibitor with a lethal dose of LPS also protected the majority of mice. Protection against LPS was seen when the inhibitor was given to mice 1 h before the LPS challenge. Furthermore, the inhibitor significantly suppressed the induction of fever by simultaneous administration with LPS without prior mixing. These results suggest that the inhibitor may be a useful potent blocker of bacterial endotoxin.

Sensitization of alveolar macrophages to lipopolysaccharide-induced prostaglandin synthesis by exogenous prostaglandins

Rabbit alveolar macrophages were found to produce extraordinary amounts of prostaglandin E2 and F2 alpha with the stimulation of lipopolysaccharide or lipid A. Exogenous prostaglandin E2 greatly enhanced the lipopolysaccharide action on rabbit alveolar macrophages for the induction of prostaglandin F2 alpha release (3-5 fold), while prostaglandin E2 alone did not cause any effect. The enhancement expressed was especially strong when prostaglandin E2 was administered to the cells simultaneously with lipopolysaccharide. The effect of prostaglandin E2 was observed neither with a nonstimulating dose of lipopolysaccharide nor with a stimulating dose of zymosan. This phenomenon was even more pronounced when prostaglandin I2 was used instead of prostaglandin E2, while no sensitization was demonstrated by prostaglandin F2 alpha. These observations suggest that prostaglandins can modulate the activation of the cyclooxygenase pathway of arachidonate metabolism in the activated macrophages by lipopolysaccharide.