TY - JOUR
T1 - Diverging pathways for lipopolysaccharide and CD14 in human monocytes
AU - Deutsch, Mordechai
AU - Kaufman, Menachem
AU - Shapiro, Howard
AU - Zurgil, Naomi
PY - 2000/12/1
Y1 - 2000/12/1
N2 - 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.
AB - 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.
KW - CD14
KW - Internalization
KW - LPS
KW - Monocyte
UR - http://www.scopus.com/inward/record.url?scp=0034548623&partnerID=8YFLogxK
U2 - 10.1002/1097-0320(20001201)41:4<279::aid-cyto6>3.0.co;2-b
DO - 10.1002/1097-0320(20001201)41:4<279::aid-cyto6>3.0.co;2-b
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
C2 - 11084613
AN - SCOPUS:0034548623
SN - 0196-4763
VL - 41
SP - 279
EP - 288
JO - Cytometry
JF - Cytometry
IS - 4
ER -