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Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on
Abstract:
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.
Insights
The Toll-like receptor 4 (TLR4)-MD-2 complex is expressed on macrophages and signals lipopolysaccharide (LPS) presence in vivo. A novel antibody inhibited LPS-induced signaling, confirming the complex
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The Toll-like receptor 4 (TLR4)-MD-2 complex is crucial for sensing lipopolysaccharide (LPS), a component of Gram-negative bacteria.
- While the human TLR4-MD-2 complex's role in LPS signaling is known, its in vivo expression and function remain largely uncharacterized.
Purpose of the Study:
- To investigate the in vivo expression and function of the mouse TLR4-MD-2 complex in response to LPS.
- To develop and utilize a specific monoclonal antibody (mAb) for studying TLR4-MD-2 interactions.
Main Methods:
- Molecular cloning of mouse MD-2.
- Development of a selective monoclonal antibody (mAb) MTS510 against the mouse TLR4-MD-2 complex.
- Flow cytometry analysis to assess cell surface expression.
- Measurement of LPS-induced NF-kappaB activation and TNF-alpha production in peritoneal macrophages.
Main Results:
- The mAb MTS510 selectively recognized the mouse TLR4-MD-2 complex but not TLR4 alone.
- Mouse MD-2 expression enhanced LPS-induced NF-kappaB activation, which was inhibited by MTS510.
- The TLR4-MD-2 complex was expressed on thioglycolate-elicited peritoneal macrophages and rapidly downregulated upon LPS exposure.
- MTS510 inhibited LPS-induced TNF-alpha production by peritoneal macrophages.
Conclusions:
- The TLR4-MD-2 complex is expressed on macrophages in vivo.
- This complex plays a significant role in sensing and signaling the presence of LPS.
- The developed mAb MTS510 is a valuable tool for studying TLR4-MD-2-mediated innate immune responses.