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Diverging pathways for lipopolysaccharide and CD14 in human monocytes
P Antal-Szalmás1, M J Poppelier, R Broekhuizen
1Eijkman-Winkler Institute, Department of Inflammation, University Medical Center, Utrecht, The Netherlands.
Cytometry
|November 21, 2000
Summary
Human monocytes possess a large intracellular CD14 pool, distinct from lipopolysaccharide (LPS) binding. LPS and CD14 internalize independently, suggesting separate clearance pathways.
Area of Science:
- Immunology
- Cell Biology
Background:
- CD14 is the primary lipopolysaccharide (LPS) binding molecule on human monocytes.
- CD14 initiates cellular responses to LPS, but its role in LPS clearance is unclear.
Purpose of the Study:
- To investigate the internalization mechanisms of LPS and CD14 on human monocytes.
- To understand the intracellular fate of LPS and CD14 following cell surface binding.
Main Methods:
- Utilized flow cytometry, trypan blue quenching, and confocal fluorescence microscopy to track FITC-LPS and CD14 uptake.
- Employed surface-biotinylation and cell subfractionation to analyze CD14 internalization.
- Quantified intracellular CD14 using enzyme-linked immunosorbent assay (ELISA).
Main Results:
- LPS internalization occurred at 1% of bound endotoxin per minute, without concurrent CD14 surface decrease.
- Demonstrated a significant intracellular CD14 pool (2.68 x 10^6 molecules/monocyte).
- Internalized LPS and CD14 were localized to distinct intracellular compartments, and CD14 was not actively internalized.
Conclusions:
- Monocytes harbor a substantial intracellular CD14 pool with an unidentified function.
- LPS and CD14 molecules can internalize independently after binding to the monocyte surface.