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Transfer of monomeric endotoxin from MD-2 to CD14: characterization and functional consequences
Athmane Teghanemt1, Polonca Prohinar, Theresa L Gioannini
1Department of Internal Medicine and the Inflammation Program, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City 52242, USA.
Abstract:
Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin depends on sequential transfer of monomers of endotoxin from an aggregated form to CD14 via the lipopolysaccharide-binding protein and then to MD-2. We now show that monomeric endotoxin can be transferred in reverse from MD-2 to CD14 but not to lipopolysaccharide-binding protein. Reverse transfer requires an approximately 1000-fold molar excess of CD14 to endotoxin-MD-2. Transfer of endotoxin from MD-2 to extracellular soluble CD14 reduces activation of cells expressing TLR4 without MD-2. However, transfer of endotoxin from MD-2 to membrane CD14 (mCD14) makes cells expressing MD-2.TLR4 sensitive to activation by the endotoxin-MD-2 complex. An endotoxin-mutant (F126A) MD-2 complex that does not activate cells expressing TLR4 alone potently activates cells expressing mCD14, MD-2, and TLR4 by transferring endotoxin to mCD14, which then transfers endotoxin to endogenous wild-type MD-2.TLR4. These findings describe a novel pathway of endotoxin transfer that provides an additional layer of regulation of cell activation by endotoxin.
Insights
Endotoxin transfer to Toll-like receptor 4 (TLR4) involves a novel reverse pathway from MD-2 to CD14. This discovery reveals new regulatory mechanisms controlling endotoxin-induced cell activation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Toll-like receptor 4 (TLR4)-mediated cell activation by endotoxin is crucial in innate immunity.
- This activation typically involves sequential transfer of endotoxin monomers from aggregated forms to CD14, then to MD-2.
Purpose of the Study:
- To investigate the potential for reverse transfer of endotoxin from MD-2 to other molecules.
- To elucidate novel pathways regulating endotoxin-induced cell activation.
Main Methods:
- Investigated endotoxin transfer dynamics using purified proteins and cell-based assays.
- Utilized mutant endotoxin-MD-2 complexes to probe specific transfer interactions.
- Quantified cell activation responses under varying concentrations of CD14 and MD-2.
Main Results:
- Demonstrated reverse transfer of monomeric endotoxin from MD-2 to CD14, but not lipopolysaccharide-binding protein.
- Showed that transfer to soluble CD14 reduces cell activation, while transfer to membrane CD14 enhances it.
- Identified a novel activation pathway involving endotoxin transfer from MD-2 to membrane CD14, then to endogenous MD-2.TLR4.
Conclusions:
- Discovered a novel reverse endotoxin transfer pathway from MD-2 to CD14.
- This pathway provides an additional regulatory mechanism for TLR4-dependent cell activation by endotoxin.
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