Analysis of HDL binding to cells.

(A) Inhibition of T-cell signaling by binding of HDL to membranes of stimulated HUT-78 cells; either membranes of stimulated HUT-78 cells (white columns), THP-1 cells (hatched columns), or both (black columns) were preincubated in the absence (−) or presence of FCS (10%), HS (10%), or HDL (0.32 mg/mL protein) for 45 minutes on ice; after washing treated (hatched and black columns) and untreated (white columns) THP-1 cells were cultured in the presence of treated (white and black columns) or untreated (hatched columns) membranes of stimulated HUT-78 cells; TNF-α (top) and IL-1β (bottom) production was measured in 48-hour culture supernatants. Results are expressed as percentage, 100% (dashed line) being the production of IL-1β or TNF-α measured in the absence of inhibitor, mean ± SD, n = 6. (B-F) Binding of unconjugated FITC and FITC-HDL (0.1 mg/mL) was assessed by flow cytometry on THP-1 cells (B), isolated human monocytes (C), unstimulated HUT-78 cells (D), and stimulated HUT-78 cells (E). FITC was used as a negative control. (F) Binding of FITC-HDL (10 μg/mL) to stimulated HUT-78 cells in the presence or absence of purified anti–apo A-I antibodies (100 μg/mL).