Diverging pathways for lipopolysaccharide and CD14 in human monocytes

Background: CD14 is considered to be the major endotoxin (lipopolysaccharid e [LPS]) binding molecule on human monocytes. It initiates cellular respons e, but its role in the clearance of LPS is not well understood. Under condi tions that ensure totally CD14-dependent LPS binding on human monocytes, th e internalization mechanisms of LPS and CD14 were studied. Methods: The uptake and intracellular distribution of fluorescein isothiocy anate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue que nching, and confocal fluorescence microscopy. Incubation of surface-biotiny lated cells with LPS at 37 degreesC or 4 degreesC and subsequent subfractio nation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent a ssay (ELISA). Results: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decre ase at the same time surface. We proved the presence of an intracellular CD 14 pool (2.68 x 10(6) molecules per unstimulated monocyte) and could show t hat internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monoc ytes showed no change in biotinylated CD14 in the membrane fraction indepen dently of the incubation temperature (37 degreesC or at 4 degreesC) used in dicating 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 CD 14 molecules can be internalized independently after association on the cel l surface. Cytometry 41:279-288, 2000. (C) 2000 Wiley-Liss, Inc.