Mononuclear cell line THP-1 internalizes bactericidal/permeability-increasing protein by a non-receptor-mediated mechanism consistent with pinocytosis
R. J. Burnett, C. A. Lyden, C. J. Tindal, C. M. Cave, M. N. Marra and J. S. Solomkin
Department of Surgery, University of Cincinnati, Ohio, College of Medicine, USA.
BACKGROUND: Bactericidal/permeability-increasing protein (BPI) binds
lipopolysaccharide and neutralizes its toxic effects in vitro and in
endotoxemic animals. Our recent work identified physiologically significant
interactions between BPI, lipopolysaccharide, and mononuclear cells.
OBJECTIVE: To determine whether the interaction between BPI and mononuclear
cells is receptor mediated. DESIGN: Labeled BPI was incubated with THP-1
cells in the presence of up to 100-fold excess of unlabeled BPI. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting
were performed to evaluate competitive binding and total uptake of BPI.
Crosslinking was performed to determine whether BPI binds to a single
protein entity. Acid washing experiments and flow cytometric analysis were
performed to determine whether BPI remains on the cellular surface.
Finally, flow cytometry analysis was used to determine whether BPI
incubation with THP-1 cells affects the surface expression of the
lipopolysaccharide-binding protein-lipopolysaccharide receptor CD14.
RESULTS: Labeled BPI uptake was not inhibited by the presence of 100-fold
excess of unlabeled BPI at 37 degrees C or 4 degrees C in the presence of
azide. Uptake was not saturable under either condition with incubation
concentrations up to 10 microgram/mL. Cross-linking did not show BPI bound
to a single entity. Acid washing and flow cytometry experiments disclosed
rapid internalization of BPI. Finally, BPI uptake by THP-1 cells had no
effect on the surface expression of CD14. CONCLUSIONS:
Bactericidal/permeability-increasing protein is rapidly internalized by
mononuclear cells in a nonspecific fashion not saturable at very high
doses, which is consistent with pinocytosis. This process may represent a
disposal mechanism for lipopolysaccharide in closed-space infections and
may be partially responsible for the rapid clearance of BPI from the
peripheral circulation.
