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 surface-biotinylated cells with LPS at 37 degrees C or 4 degrees 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 CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10(6) 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 degrees C or at 4 degrees 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.
Antal-Szalmás
P
P
Eijkman-Winkler Institute, Department of Inflammation, University Medical Center, Utrecht, The Netherlands.
Poppelier
M J
MJ
Broekhuizen
R
R
Verhoef
J
J
van Strijp
J A
JA
van Kessel
K P
KP
D019921:Biotinylation; D002458:Cell Fractionation; D002478:Cells, Cultured; D015536:Down-Regulation; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000032:analysis / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D014343:Trypan Blue / Q000378:metabolism
D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016650:Fluorescein-5-isothiocyanate; D014343:Trypan Blue