Figure 6.

SR-BI-mediated internalization of Hb-apoAI complex. (A) Quantification of the internalization of SR-BI, metHb and apoAI into hepatocytes (HepG2) and macrophages (THP-1). The % endocytosed proteins were normalized against either total biotinylated cell surface expression for the SR-BI receptor or total biotinylated proteins applied (metHb, apoAI); * indicates the internalized SR-BI, metHb or apoAI; ◄ indicates the internalization level upon treatment with 0.45 M sucrose. Two receptors, TFR and ABCA1, served as negative controls in the cells incubated with metHb and/or apoAI. (B) Immunofluorescence microscopy of internalized metHb-apoAI. The intracellular white spots in the merged images indicate co-localized proteins. (C) Cytokine production by HepG2 and THP-1 following endocytosis of metHb-apoAI. Increasing amounts of proteins (metHb and/or apoAI) were incubated with the cells for 1 h to allow endocytosis to occur. The production of TNF-α and IL-6 in the cell supernatant was determined after 24 h. The red boxes highlight the dose-dependent upregulation of TNF-α and IL-6 upon endocytosis of metHb-apoAI complex. (D) A model to illustrate the antioxidant activities of lipid-free apoAI and Hp. Cell-free oxidative Hb was rapidly captured by Hp and apoAI to form complexes in plasma, all of which quenched the redox activity of Hb (Hb^POX). Following association with Hb, the apoAI further leads the internalization of Hb-apoAI complex via its recognition by SR-BI on macrophages and hepatocytes. Black color represents interactions identified in this study while grey indicates either known or speculated process.