Abstract
When bacterial lipopolysaccharide (LPS) enters the bloodstream, it is thought to have two general fates. If LPS binds to circulating leukocytes, it triggers innate host defense mechanisms and often elicits toxic reactions. If instead LPS binds to plasma lipoproteins, its bioactivity is largely neutralized. This study shows that lipoproteins can also take up LPS that has first bound to leukocytes. When monocytes were loaded with [(3)H]LPS and then incubated in plasma, they released over 70% of the cell-associated [(3)H]LPS into lipoproteins (predominantly high density lipoprotein), whereas in serum-free medium the [(3)H]LPS remained tightly associated with the cells. The transfer reaction could be reproduced in the presence of pure native lipoproteins or reconstituted high density lipoprotein. Plasma immunodepletion experiments and experiments using recombinant LPS transfer proteins revealed that soluble CD14 significantly enhances LPS release from the cells, high concentrations of LPS-binding protein have a modest effect, and phospholipid transfer protein is unable to facilitate LPS release. Essentially all of the LPS on the monocyte cell surface can be released. Lipoprotein-mediated LPS release was accompanied by a reduction in several cellular responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo.
Highlights
Gram-negative bacterial lipopolysaccharide (LPS)1 is one of the most potent and ubiquitous of the known bacterial signal molecules
Two plasma lipid transfer proteins, LPS-binding protein (LBP) [18, 19] and phospholipid transfer protein (PLTP) [20], promote the binding of purified LPS to lipoproteins, whereas only LBP can facilitate LPS binding to CD14 on cell membranes or soluble CD14 in plasma [1, 21, 22]. sCD14 can rapidly transfer LPS to mCD14 on cells [23], and it facilitates the activation of cells that do not express mCD14 [24]
We found that after exposure to only 3 ng/ml [3H]LPS, the percentage of cell-associated LPS released in the presence of nHDL and sCD14 in 1 h was only slightly greater than the percentage of LPS released by cells that had been exposed to 50 ng/ml [3H]LPS
Summary
Cells and Reagents—Human monocytic THP-1 cells were cultured as described previously [26]. The incubations were stopped with 0.3 ml of cold PBS, the cells were removed by centrifugation, and the radioactivity in the cells and supernatants was counted to measure bound and released [3H]LPS. A significant degree of membrane permeability (Ͼ10% permeable cells) was observed only in plasma samples that contained anti-PLTP or high concentrations of rsCD14; cell permeability induced by these reagents was completely inhibited by EDTA or by removal of plasma. The cells were incubated in 100 l of SFM (4 ϫ 106 THP-1 cells/ml) or RPMI 1640 medium containing 10 mM HEPES, pH 7.4, and 0.1 mg of bovine serum albumin/ml (7–12 ϫ 106 PBMC/ml) in microcentrifuge tubes or 96-well culture plates, respectively. The cytokines were measured by ELISA using DuoSet kits or match antibody pairs from Genzyme, Cambridge, MA (IL-8, IL-1, and IL-6) or Pharmingen (TNF-␣)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
