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

Apoptosis assays with lymphoma cell lines: problems and pitfalls

Much attention has been focused on the manner in which tumour cells die after treatment with cytotoxic agents. The basic question is whether cells die via apoptosis or via direct damage from the toxic agent. Various assays have been used to make this distinction. However, we show herein that some of the widely used assays for apoptosis do not in fact distinguish between apoptosis and other forms of cell death. More specifically: (1) A sub-G1 DNA content, identified by propidium iodide staining, does not distinguish between apoptotic and necrotic cells; (2) loss of mitochondrial membrane potential does not distinguish between apoptotic and necrotic cells, unless combined with an assay for an intact cell membrane; (3) subcellular fragments that arise from dead cells or from apoptotic bodies can interfere with some assays for apoptosis such as annexin V staining, as they may be close to the size of intact cells, making it difficult to decide where to set the size threshold; (4) irradiated cells display a large increase in nonspecific Ab binding. This may be partly due to an increase in cell size, but, regardless of the cause, it can lead to a mistaken conclusion that there is an increase in a particular antigen if appropriate control reagents are not tested; and (5) experiments utilising Ab crosslinking have neglected the role of cell aggregation, which can cause multiple problems including death from mechanical stress when cells are handled. Consideration of these factors will improve our ability to determine the mode of cell death

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1–2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes

Pb(II) Induces Scramblase Activation and Ceramide-Domain Generation in Red Blood Cells

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.AGA was a predoctoral student supported by the Basque Government and later by the University of the Basque Country (UPV/EHU). This work was also supported in part by grants from the Spanish Government (FEDER/MINECO BFU 2015-66306-P to F.M.G. and A.A.) and the Basque Government (IT849-13 to F.M.G. and IT838-13 to A.A.), and by the Swiss National Science Foundation

Nanoelectropulse-driven membrane perturbation and small molecule permeabilization

BACKGROUND: Nanosecond, megavolt-per-meter pulsed electric fields scramble membrane phospholipids, release intracellular calcium, and induce apoptosis. Flow cytometric and fluorescence microscopy evidence has associated phospholipid rearrangement directly with nanoelectropulse exposure and supports the hypothesis that the potential that develops across the lipid bilayer during an electric pulse drives phosphatidylserine (PS) externalization. RESULTS: In this work we extend observations of cells exposed to electric pulses with 30 ns and 7 ns durations to still narrower pulse widths, and we find that even 3 ns pulses are sufficient to produce responses similar to those reported previously. We show here that in contrast to unipolar pulses, which perturb membrane phospholipid order, tracked with FM1-43 fluorescence, only at the anode side of the cell, bipolar pulses redistribute phospholipids at both the anode and cathode poles, consistent with migration of the anionic PS head group in the transmembrane field. In addition, we demonstrate that, as predicted by the membrane charging hypothesis, a train of shorter pulses requires higher fields to produce phospholipid scrambling comparable to that produced by a time-equivalent train of longer pulses (for a given applied field, 30, 4 ns pulses produce a weaker response than 4, 30 ns pulses). Finally, we show that influx of YO-PRO-1, a fluorescent dye used to detect early apoptosis and activation of the purinergic P2X(7 )receptor channels, is observed after exposure of Jurkat T lymphoblasts to sufficiently large numbers of pulses, suggesting that membrane poration occurs even with nanosecond pulses when the electric field is high enough. Propidium iodide entry, a traditional indicator of electroporation, occurs with even higher pulse counts. CONCLUSION: Megavolt-per-meter electric pulses as short as 3 ns alter the structure of the plasma membrane and permeabilize the cell to small molecules. The dose responses of cells to unipolar and bipolar pulses ranging from 3 ns to 30 ns duration support the hypothesis that a field-driven charging of the membrane dielectric causes the formation of pores on a nanosecond time scale, and that the anionic phospholipid PS migrates electrophoretically along the wall of these pores to the external face of the membrane

Tropical limestone forest resilience and late Pleistocene foraging during MIS‐2 in the Tràng an Massif, Vietnam

In this paper we present a multi‐proxy study of tropical limestone forest and its utilization by human groups during the major climatic and environmental upheavals of MIS‐2 (29‐11.7 kBP). Our data are drawn from new field research within the Tràng An World Heritage property on the edge of the Red River Delta, northern Vietnam. Key findings from this study include 1) that limestone forest formations were resilient to the large‐scale landscape transformation of the Sunda continent at the end of the last glaciation; 2) that prehistoric human groups were probably present in this habitat through‐out MIS‐2; and 3) that the forested, insular, karst of Tràng An provided foragers with a stable resource‐base in a wider changing landscape. These results have implications for our understanding of the prehistoric utilization of karst environments and resonance for their conservation in the face of climate and environmental change today

Phosphatidylserine targeting for diagnosis and treatment of human diseases

Cells are able to execute apoptosis by activating series of specific biochemical reactions. One of the most prominent characteristics of cell death is the externalization of phosphatidylserine (PS), which in healthy cells resides predominantly in the inner leaflet of the plasma membrane. These features have made PS-externalization a well-explored phenomenon to image cell death for diagnostic purposes. In addition, it was demonstrated that under certain conditions viable cells express PS at their surface such as endothelial cells of tumor blood vessels, stressed tumor cells and hypoxic cardiomyocytes. Hence, PS has become a potential target for therapeutic strategies aiming at Targeted Drug Delivery. In this review we highlight the biomarker PS and various PS-binding compounds that have been employed to target PS for diagnostic purposes. We emphasize the 35 kD human protein annexin A5, that has been developed as a Molecular Imaging agent to measure cell death in vitro, and non-invasively in vivo in animal models and in patients with cardiovascular diseases and cancer. Recently focus has shifted from diagnostic towards therapeutic applications employing annexin A5 in strategies to deliver drugs to cells that express PS at their surface