Rintaro Shimazu

Cutting Edge: Cell Surface Expression and Lipopolysaccharide Signaling Via the Toll-Like Receptor 4-MD-2 Complex on Mouse Peritoneal Macrophages

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-κB 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-α 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.

Mouse Toll-like Receptor 4·MD-2 Complex Mediates Lipopolysaccharide-mimetic Signal Transduction by Taxol

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 TLR4·MD-2 complex mediates a Taxol-induced 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 NFκB activation in these transfectants. Noticeable NFκB 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 TLR4·MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFκB activation via TLR4·MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFκB activation via TLR4·MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4·MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.

Involvement of TLR4/MD-2 complex in species-specific lipopolysaccharide-mimetic signal transduction by Taxol

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice, but not in humans. 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. Here we examined whether or not TLR4/MD-2 complex mediates a Taxol-induced signal by using 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. Our results demonstrated that co-expression of mouse TLR4 and mouse MD-2 was required for Taxol responsiveness, and that the TLR4/MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice. We also found that mouse MD-2, but not human MD-2, is involved in Taxol signaling, suggesting that MD-2 is responsible for the species-specific responsiveness to Taxol.

Induction of long-term lipopolysaccharide tolerance by an agonistic monoclonal antibody to the toll-like receptor 4/MD-2 complex

ABSTRACT We have established an agonistic monoclonal antibody, UT12, that induces stimulatory signals comparable to those induced by lipopolysaccharide (LPS) through Toll-like receptor 4 and MD-2. UT12 activated nuclear factor κB and induced the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in peritoneal exudative cells. In addition, mice injected with UT12 rapidly fell into endotoxin shock concomitant with the augmentation of serum TNF-α and IL-6 levels, followed by death within 12 h. On the other hand, when the mice were pretreated with a sublethal dose of UT12, the mice survived the subsequent lethal LPS challenges, with significant suppression of serum TNF-α and IL-6, indicating that UT12 induced tolerance against LPS. This effect of UT12 was maintained for at least 9 days. In contrast, the tolerance induced by LPS continued for less than 3 days. These results illuminate a novel potential therapeutic strategy for endotoxin shock by the use of monoclonal antibodies against the Toll-like receptor 4/MD-2 complex.