Diverging pathways for lipopolysaccharide and CD14 in human monocytes

Background: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. Methods: The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surfacebiotinylated cells with LPS at 37°C or 4°C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). Results: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD 14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10⁶ molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37°C or at 4°C) used, indicating that these CD14 molecules were not taken up by an active process. Conclusions: These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface. (C) 2000 Wiley-Liss, Inc.