Activity of monosaccharide lipid A analogues in human monocytic cells as agonists or antagonists of bacterial lipopolysaccharide

Abstract

The lipid A portion of bacterial lipopolysaccharide (LPS) plays a central role in the production of endotoxic mediators. Different responses between human and murine macrophages to lipid A-like structures have been indicated. We investigated a series of structurally related monosaccharide lipid A analogues for their potency to activate human macrophage U937 cells and peripheral blood mononuclear cells for production of tumor necrosis factor-alpha and interleukin-6 compared with their potency to activate murine macrophage RAW264.7 cells. Two of the analogues were found to have sufficient potency to activate the human cells as well as the murine cells. These analogues comprise D-glucosamine, phosphoryl groups, and acyl groups of defined carbon chain lengths (C(14) and C(12)) in a ratio of 1:1:3. This ratio of molecular constituents is proportional to that of the complete disaccharide structure of lipid A (2:2:6). Other analogues with two or four C(14) acyl groups and with three acyl groups but including a C(10) or a C(16) acyl group, which are active to murine cells, showed no LPS-agonistic activity, but did show LPS-antagonistic activity, to human cells. An LPS-antagonistic analogue in the murine cells also showed antagonistic activity in human cells. These results reveal that lipid A analogues recognized as being LPS agonists by human macrophages have common structural features in monosaccharide and disaccharide structures which are more strict than those required for recognition by murine macrophages and that broad lipid A-like structures are recognized as being LPS antagonists by human cells but are recognized by murine cells as being either LPS agonists or antagonists.

FIG. 1

Induction of TNF-α and IL-6 release from murine RAW264.7 cells (A and D), human U937 cells (B and E), and human PBMC (C and F) by synthetic lipid A analogues. Murine RAW264.7 cells (5 × 10⁵ cells/0.5 ml/well of a 48-well culture plate) were cultured in 5% FCS-RPMI medium for 2 h and washed three times with Hanks' balanced salt solution. The adherent cells were cultured in 5% FCS-RPMI medium with test samples. Human U937 cells (10⁵ cells/0.5 ml/well of a 48-well culture plate) were cultured in 10% FCS-RPMI medium with 30 ng of PMA/ml for 3 days and washed once with 10% FCS-RPMI medium. The adherent cells were cultured in 10% FCS-RPMI medium with test samples. Human PBMC were isolated from heparinized peripheral blood of healthy volunteer donors by density gradient centrifugation with low-endotoxin Ficoll-Hypaque. The cells (5 × 10⁵ cells/0.5 ml/well of a 48-well culture plate) were cultured in 10% FCS-RPMI medium with test samples. Culture supernatant from these three different types of cells obtained at 4 and 24 h after stimulation with test samples at the indicated concentrations was assayed for TNF-α (A, B, and C) and IL-6 (D, E, and F), respectively. Synthetic lipid A analogues tested were 506 with a hexacylated disaccharide structure (complete lipid A), 406 with a tetra-acylated disaccharide structure, and GLA-60 with a triacylated monosaccharide structure. Chemical structures of these analogues are shown at the top. Data are the means ± SEM of triplicate samples. A representative result of three independent experiments is shown.

FIG. 2

Effect of the number of attached acyl groups in monosaccharide lipid A analogues on induction of TNF-α and IL-6 release from murine RAW264.7 cells (A and D), human U937 cells (B and E), and human PBMC (C and F). Preparation and culture of cells were as described in the legend for Fig. 1. These cells were stimulated with monosaccharide lipid A analogues GLA-26 (two acyls), GLA-60 (three acyls), and GLA-47 (four acyls) at the indicated concentrations for 4 and 24 h, and the culture supernatant was assayed for TNF-α (A, B, and C) and IL-6 (D, E, and F), respectively. Chemical structures of these analogues are shown at the top. Data are the means ± SEM of triplicate samples. Similar results were obtained in another experiment.

FIG. 3

Effect of the carbon chain length of a secondary acyl group in triacylated monosaccharide lipid A analogues on induction of TNF-α release from murine RAW264.7 cells (A) and human U937 cells (B). Preparation and culture of the cells were as described in the legend for Fig. 1. These cells were stimulated for 4 h with monosaccharide lipid A analogues of differing carbon chain lengths in their branched acyl side chains at the indicated concentrations, and the supernatants were assayed for TNF-α. As shown in the structures, GLA-89, GLA-63, GLA-60, and GLA-64 have secondary acyl groups with carbon chain lengths of C₁₀, C₁₂, C₁₄, and C₁₆, respectively. Data are the means ± SEM of triplicate samples. A representative result of three independent experiments is shown.

FIG. 4

Inhibitory effect of synthetic lipid A analogues on LPS-induced TNF-α and IL-6 release from human U937 cells. U937 cells were cultured in the presence of synthetic lipid A analogues at the indicated concentrations for 30 min and then stimulated with LPS at 10 ng/ml. Culture supernatant obtained at 4 and 24 h after LPS stimulation was assayed for TNF-α (A) and IL-6 (B), respectively. Lipid A analogues tested were 406 (○), GLA-26 (▴), GLA-47 (▵), GLA-89 (□), and GLA-64 (■). The levels of TNF-α and IL-6 production due to LPS stimulation alone in the absence of these analogues (gray bands) are (means ± SEM) 178 ± 46 and 160 ± 30 U/ml, respectively. Data are the means ± SEM of triplicate samples. A representative result of three independent experiments is shown.

FIG. 5

Antagonistic action of monosaccharide lipid A analogue GLA-58 in human U937 cells. U937 cells were cultured in the presence of GLA-58 at the indicated concentrations for 30 min and then stimulated with LPS at 10 ng/ml. Culture supernatant obtained at 4 and 24 h after LPS stimulation was assayed for TNF-α (A) and IL-6 (B), respectively. The levels of TNF-α and IL-6 production by LPS stimulation alone in the absence of GLA-58 are shown as gray bands. The structure of GLA-58 is shown. Data are the means ± SEM of triplicate samples. A representative result of three independent experiments is shown.

FIG. 6

Structures of synthetic lipid A analogues in relation to their LPS-agonistic and -antagonistic actions in murine and human macrophages (Mφ).