Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.

The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4⅐MD-2 complex mediates a Taxolinduced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFB activation in these transfectants. Noticeable NFB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4⅐MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFB activation via TLR4⅐MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFB activation via TLR4⅐MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4⅐MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.Taxol, a diterpene purified from the bark of the Western yew (Taxus brevifolia) (1), is an antitumor agent that blocks mitosis by binding and stabilizing microtubules (2, 3). Ding et al. (4) found that Taxol induces the secretion of tumor necrosis factor and down-regulation of tumor necrosis factor receptors in murine macrophages. Although the structure of Taxol is quite different from that of lipopolysaccharide (LPS), 1 Taxol has been shown to possess many LPS-like activities, such as tyrosine phosphorylation of microtubule-associated protein kinases (5), induction of LPS-inducible gene expression (6), and activation of NFB (7). Interestingly, Taxol mimics the actions of LPS on murine macrophages but not on human LPS-responsive cells including macrophages (8,9).LPS contains polysaccharide and lipid A portion, the latter of which mediates many LPS responses (10). Several synthetic and natural lipid A analogs, which lack LPS-like activities, have been shown to retain the ability to block LPS-induced cellular responses (11-13). Taxol-induced signaling events in murine macrophages are blocked by some of these LPS antagonists, suggesting that LPS and Taxol share a receptor or signaling molecule (14). Although the target of the antagonists was not defined well, membrane-bound CD14 (mCD14), which has been demonstrated to be involved in LPS-induced signaling events on macrophages (15), might not be the target, because the ...

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