Abstract
A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213-12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin.TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor alpha production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor alpha production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.
Highlights
Flagellin, the major structural protein of flagella from Gramnegative bacteria, is an extraordinarily potent activator of inflammatory cells such as monocytes, macrophages, epithelial cells, osteoblasts, and fibroblasts [1,2,3,4,5,6,7,8,9,10,11,12,13,14]
Flagellin Binds to Surface-associated toll-like receptor 5 (TLR5) in RAW 264.7 Cells—As a first step in the evaluation of the action of gangliosides on flagellin signaling, we developed an assay for TLR5dependent flagellin binding to cells
Gangliosides Inhibit Flagellin-induced TNF␣ Production in HeNC2, THP-1, and RAW 264.7 Cells—After establishing an assay for the cell surface binding of flagellin to TLR5 (Fig. 1) and that TLR5 signaling occurs at the cell surface, we focused our analysis on the question of gangliosides as co-receptors for TLR5
Summary
Cells and Reagents—The C3H/HeN-derived macrophage cell line HeNC2 and the human monocytic cell line THP-1 were maintained in RPMI 1640 containing 10% fetal bovine serum. Endotoxin-depleted recombinant Histagged S. enteritidis, P. aeruginosa (PAO1), and Escherichia coli (EPEC) flagellins were prepared as described previously [2] None of these flagellins exhibit stimulatory activity on TLR5-negative RAW 264.7 cells, indicating that the level of potential contaminants such as lipopolysaccharide are well below the range necessary for bioactivity. The effect of PPMP on membrane ganglioside expression was evaluated by measuring the level of Alexa fluor 488 cholera toxin subunit B conjugate binding to RAW 264.7 cells incubated in the presence or absence of PPMP. To assess the effect of gangliosides on TNF␣ production in TLR5-EYFP-expressing RAW 264.7 cells, the cells (8 ϫ 104 cells/well) were cultured overnight in 12-well plates in serum-free medium with or without flagellin and 10 g/ml gangliosides. The culture medium from each well was harvested and analyzed for TNF␣ content by enzyme-linked immunosorbent assay
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