Mononuclear Cell Line THP-1 Internalizes Bactericidal/Permeability-Increasing Protein by a Non—Receptor-Mediated Mechanism Consistent With Pinocytosis
Robert J. Burnett, MD;
Catherine A. Lyden;
Carolyn J. Tindal, MS;
Cynthia M. Cave;
Marian N. Marra, MA;
Joseph S. Solomkin, MD
Arch Surg. 1996;131(2):200-206.
Abstract
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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. Cross-linking 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°C or 4°C in the presence of azide. Uptake was not saturable under either condition with incubation concentrations up to 10 µg/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 CD 14.
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.
(Arch Surg. 1996;131:200-206)
Author Affiliations
From the Department of Surgery, University of Cincinnati (Ohio) College of Medicine (Drs Burnett and Solomkin and Mss Lyden, Tindal, and Cave); and Incyte Pharmaceuticals Inc, Palo Alto, Calif (Ms Marra).
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