Original Article

The identification of specific high density lipoprotein₃ binding sites on human blood monocytes using fluorescence-labeled ligand

Correspondence to: Hiroya Hidaka

We previously reported the identity and purification of two HDL₃-binding proteins in rat liver plasma membranes. As these proteins are candidate high density lipoprotein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying corresponding proteins that are present in human tissue. This report describes the identification of HDL₃-binding sites on human monocytes with the use of fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)-labeled human HDL₃, fluorescence micrographs showed dense signals of fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL₃ and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL₂ only partially competed for binding, a 50-fold concentration of HDL₃ did compete effectively for binding of FITC-HDL₃ to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FITC-HDL₃ remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL₃ as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB₁ and HB₂ from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.—Hidaka, H., E. Hidaka, M. Tozuka, J. Nakayama, T. Katsuyama, and N. Fidge. Identification of specific high density lipoprotein₃ binding sites on human blood monocytes using fluorescence-labeled ligand. J. Lipid Res. 1999. 40: 1131–1139.

Supplementary key words: HDL₃, HDL-binding protein, monocyte, flow cytometry, fluorescent isothiocyanate

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