Transbilayer phospholipid movements in ABCA1-deficient cells.

Tangier disease is an inherited disorder that results in a deficiency in circulating levels of HDL. Although the disease is known to be caused by mutations in the ABCA1 gene, the mechanism by which lesions in the ABCA1 ATPase effect this outcome is not known. The inability of ABCA1 knockout mice (ABCA1-/-) to load cholesterol and phospholipids onto apoA1 led to a proposal that ABCA1 mediates the transbilayer externalization of phospholipids, an activity integral not only to the formation of HDL particles but also to another, distinct process: the recognition and clearance of apoptotic cells by macrophages. Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient engulfment of the apoptotic cells, and it has been proposed that ABCA1 is required for transbilayer externalization of PS to the surface of both cell types. To determine whether ABCA1 is responsible for any of the catalytic activities known to control transbilayer phospholipid movements, these activities were measured in cells from ABCA1-/- mice and from Tangier individuals as well as ABCA1-expressing HeLa cells. Phospholipid movements in either normal or apoptotic lymphocytes or in macrophages were not inhibited when cells from knockout and wildtype mice or immortalized cells from Tangier individuals vs normal individuals were compared. Exposure of PS on the surface of normal thymocytes, apoptotic thymocytes and elicited peritoneal macrophages from wildtype and knockout mice or B lymphocytes from normal and Tangier individuals, as measured by annexin V binding, was also unchanged. No evidence was found of ABCA1-stimulated active PS export, and spontaneous PS movement to the outer leaflet in the presence or absence of apoA1 was unaffected by the presence or absence of ABCA1. Normal or Tangier B lymphocytes and macrophages were also identical in their ability to serve as targets or phagocytes, respectively, in apoptotic cell clearance assays. No evidence was found to support the suggestion that ABCA1 is involved in transport to the macrophage cell surface of annexins I and II, known to enhance phagocytosis of apoptotic cells. These results show that mutations in ABCA1 do not measurably reduce the rate of transbilayer movements of phospholipids in either the engulfing macrophage or the apoptotic target, thus discounting catalysis of transbilayer movements of phospholipids as the mechanism by which ABCA1 facilitates loading of phospholipids and cholesterol onto apoA1.

Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes.

When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macrophages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The inhibition is not additive, suggesting that the exposed PS molecules on the two cells participate in a common process. We asked whether this dual requirement reflects bridging of the target cell and macrophage by bivalent, PS-binding annexins. Monoclonal antibodies (mAbs) against annexins I or II stained a variety of live phagocytes. Apoptotic Jurkat T lymphocytes and human peripheral T lymphocytes, but not apoptotic thymocytes, were stained by anti-annexin I but not II. Phagocytosis of apoptotic targets was inhibited by mAbs to annexins I or II, or by pretreatment of macrophages with the same mAbs. Pretreatment of apoptotic thymocytes had no effect, whereas pretreating Jurkat cells with anti-annexin I or removing annexin I with EGTA was inhibitory. Annexin bridging is vectorial, because annexin is bound to PS molecules on targets but not on macrophages, suggesting annexins serve as both ligand and receptor in promoting phagocytosis.

Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells.

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human monocytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocytosis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating monocytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis.