Cell activation by monosaccharide lipid A analogues utilizing Toll-like receptor 4

Abstract

Lipid A is the bioactive centre of lipopolysaccharide (LPS), and its properties exhibit various endotoxic and biological effects toward host cells. We examined whether Toll-like receptors (TLRs) are mediated by the signals from various synthetic acylated derivatives of d-glucosamine monophosphate. All test synthetic monosaccharide lipid A analogues similar to acylated beta-(1-6)-d-glucosamine disaccharide bisphosphates, such as Escherichia coli-type lipid A (compound 506) and its precursor (compound 406), clearly induced nuclear factor (NF)-kappaB activation in Ba/F3 cells expressing murine TLR4 and its accessory protein MD-2 (Ba/mTLR4/mMD-2), but no induction was found in those expressing murine TLR2 (Ba/mTLR2). Compound 411, the non-reducing sugar moiety of compound 506, exhibited interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha)-producing activities in human peripheral blood mononuclear cells (PBMC), whereas compound 401, the reducing moiety of compounds 506 and 406, and Gifu lipid A-46 (GLA-46), the non-reducing moiety of compound 406, induced no production of IL-8 and TNF-alpha, which was similar to the findings for compound 406. Among the synthetic triacylated monosaccharide lipid A analogues, some compounds with three tetradecanoyl (C14) groups or that included a dodecanoyl (C12) group were more active toward murine and human cells than were other analogues with a decanoyl (C10) or hexadecanoyl (C16) group. Furthermore, IL-8 production in PBMC stimulated with the active monosaccharide lipid A analogues as well as compound 506 was clearly inhibited by the monoclonal antibody to human TLR4. These findings suggest that monosaccharide lipid A analogues similar to disaccharide lipid As are capable of activating both murine and human cells through TLR4.

Figure 1

Chemical structures of synthetic lipid A and its analogue. Abbreviations used: C₁₄-OH, (R)-3-hydroxytetradecanoyl; C₁₄-O-(C₁₂), (R)-3-dodecanoyloxytetradecanoyl; C₁₄-O-(C₁₄), (R)-3-tetradodecanoyloxytetradecanoyl.

Figure 2

Chemical structures of synthetic acylated glucosamine monophosphates. Abbreviations used: P, PO (OH)₂; C₁₄-OH, (R)-3-hydroxytetradecanoyl; C₁₄-O-(C₁₀), (R)-3-dodecanoyloxytetradecanoyl; C₁₄-O-(C₁₂), (R)-3-dodecanoyloxytetradecanoyl; C₁₄-O-(C₁₄), (R)-3-tetradodecanoyloxytetradecanoyl; C₁₄-O-(C₁₆), (R)-3-hexadecanoyloxytetradecanoyl.

Figure 3

NF-κB activation in Ba/F3 cells in response to various synthetic disaccharide and monosaccharide lipid A analogues. Cells were stimulated with the indicated doses of each test specimen in RPMI-1640 supplemented with 10% FBS at 37° for 4 hr. Luciferase activity in the cell lysate was measured. Results are shown as relative luciferase activity, which is a ratio of stimulated activity to non-stimulated activity, in each cell line. Similar results were obtained from three independent experiments. Peptidoglycan (PGN) and TNF-α were used as positive control specimens in this experiment.

Figure 4

IL-8-producing activities in human PBMC by various synthetic disaccharide and monosaccharide lipid A analogues. Cells were stimulated with the indicated doses of each test specimen in RPMI-1640 supplemented with 10% FBS at 37° for 48 hr. After incubation, the supernatants were collected and IL-8 production was determined by ELISA. Experiments were performed at least three times and representative results are shown. Each assay was performed in triplicate wells and data are expressed as the mean ± SEM. Significant differences between groups with and without the test specimens were seen (*P < 0·01).

Figure 5

TNF-α-producing activities in human PBMC by various synthetic disaccharide and monosaccharide lipid A analogues. Cells were stimulated with the indicated doses of each test specimen in RPMI-1640 supplemented with 10% FBS at 37° for 24 hr. After incubation, the supernatants were collected and TNF-α production was determined by ELISA. Experiments were performed at least three times and representative results are shown. Each assay was performed in triplicate wells and data are expressed as the mean ± SEM. Significant differences between groups with and without the test specimens were seen (*P < 0·01).

Figure 6

Effect of mouse monoclonal antibodies to human TLR2 and TLR4 on IL-8-producing activities in PBMC stimulated with synthetic disaccharide and monosaccharide lipid A analogues. Cells were incubated with or without 10 μg/ml of mouse monoclonal antibodies to human TLR2 (TL2.1), to human TLR4 (HTA125), or mouse IgG2a at 37° for 30 min, prior to the addition of compound 506 (10 ng/ml) or various monosaccharide-type lipid A analogues (each 10 μg/ml) supplemented with 10% FBS. After 48 hr of incubation, the supernatants were collected and IL-8 production was determined by ELISA. The experimental protocols were the same as described in the legend to Figure 4. Each assay was carried out in triplicate wells and data are expressed as the mean ± SEM. Significant differences were seen as compared to the medium alone (*P < 0·01).