Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy.

Abstract

We have developed a procedure for labeling lipoproteins with the fluorescent probe 3,3'-dioctadecylindocarbocyanine (Dil) and have used Dil-labeled native and acetoacetylated lipoproteins to differentiate macrophages from fibroblasts in mixed cell culture. Lipoproteins labeled with this probe were suitable for the direct viewing of their binding and internalization by cells in vitro. The labeling technique has been applied to human low density lipoproteins (LDL) and to two canine cholesterol-induced lipoproteins: apo-E HDLc, which contain only the E apoprotein (apo-E), and beta-migrating, very low density lipoproteins (beta-VLDL), which contain apo-B and apo-E. The Dil-labeled lipoproteins showed specific high affinity binding to human fibroblasts via the LDL (apo-B, -E) receptors. The equilibrium dissociation constant for the binding of Dil-labeled apo-E HDLc and LDL were the same as for the respective native lipoproteins. The specific binding of Dil-labeled LDL and apo-E HDLc was further substantiated by fluorescence microscopy. When an excess of native (non-fluorescent) lipoproteins was added to the Dil-labeled lipoproteins, essentially no fluorescently labeled lipoproteins were seen associated with the cells. The Dil-labeled LDL, apo-E HDLc, and beta-VLDL, which were bound to the cells at 4 degrees C, were associated with the cell surface and were often observed in linear arrays. Cells that were either incubated with Dil-labeled lipoproteins at 4 degrees C and subsequently heated to 37 degrees C or incubated with the Dil-labeled lipoproteins at 37 degrees C showed internalized perinuclear fluorescence. When Dil-labeled LDL, apo-E HDLc, or beta-VLDL were treated with diketene to acetoacetylate their lysine residues, and then were incubated at 37 degrees C with mixtures of fibroblasts and mouse peritoneal macrophages in culture, the fibroblasts did not become fluorescently labeled. The macrophages became highly fluorescent, however. The acetoacetylation inhibited the interaction of the lipoproteins with the apo-B, -E receptors of fibroblasts and stimulated their uptake by macrophages. The use of fluorescently labeled native lipoproteins and chemically modified lipoproteins may allow the functional differentiation of macrophages from other cell types (e.g., fibroblasts and smooth muscle cells) in the arterial wall. This differentiation may be useful in determining the origin of the lipid-laden foam cells of atherosclerotic lesions.