Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry.
BACKGROUND
The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface. Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum.


METHODS
A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver. After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R. phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions.


RESULTS
Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process. The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding. The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations.


CONCLUSIONS
Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations. Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types.
Catalá
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M
Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense, Madrid, Spain.
Antón
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A
Portolés
M T
MT
D000818:Animals; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry*; D005434:Flow Cytometry / Q000379:methods*; D007728:Kupffer Cells / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D012772:Shock, Septic; D013997:Time Factors
D004731:Endotoxins; D008070:Lipopolysaccharides