Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages. (17/2206)

Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.  (+info)

Phosphatidylserine externalization during differentiation-triggered apoptosis of erythroleukemic cells. (18/2206)

K562 erythroleukemia cells undergo apoptosis when induced to differentiate along the erythroid lineage with hemin. This event, characterized by DNA fragmentation, correlated with downregulation of the survival protein, BCL-xL, and decrease in mitochondrial transmembrane potential (deltapsi[m]) that ultimately resulted in cell death. Reorientation of phosphatidylserine (PS) from the cells inner-to-outer plasma membrane leaflet and inhibition of the aminophospholipid translocase was observed upon hemin-treatment. Constitutive expression of BCL-2 did not inhibit hemin-induced alterations in lipid asymmetry or decrease in deltapsi[m], and only moderately prevented DNA fragmentation. BCL-2, on the other hand, effectively inhibited actinomycin D-induced DNA fragmentation, the appearance of PS at the cells outer leaflet and the decrease in deltapsi[m]. The caspase inhibitor, z.VAD.fmk, blocked DNA fragmentation by both hemin and actinomycin D, but inhibited PS externalization only in the actinomycin D-treated cells. These results suggest that, unlike pharmacologically-induced apoptosis, PS externalization triggered by differentiation-induced apoptosis occurs by a mechanism that is associated with a decrease in deltapsi[m], but independent of BCL-2 and caspases.  (+info)

Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes. (19/2206)

In lymphocytes, an asymmetric distribution of phospholipids across the plasma membrane is maintained by an ATP-dependent translocase which specifically transports aminophospholipids from the outer to the inner leaflet of the bilayer. During apoptosis, this enzyme is down-regulated and a lipid flipsite, termed the scramblase, is activated. Together, these events lead to the appearance of phosphatidylserine (PS) on the cell surface. In DO11.10 T lymphocyte hybridoma cells undergoing apoptosis, the kinetics of PS externalization are paralleled by the development of PS-sensitive phagocytosis by macrophages. This parallel is also observed when PS externalization is effected directly by application of a Ca2+ ionophore, suggesting that PS externalization is not only necessary, but sufficient, to generate a recognition signal. The broad spectrum aspartate-directed cysteine protease (caspase) inhibitor zVAD-fmk blocks externalization of PS and terminal cell lysis after induction of apoptosis by anti-CD3 antibody, but is ineffective when apoptosis is induced in the same cells by treatment with glucocorticoid. These results suggest that apoptosis induced by glucocorticoid does not require the same zVAD-sensitive caspase steps which are required for Fas/FasL-dependent death induced by anti-CD3 antibody, and that the action of these proteases is also not required for PS externalization. Extracellular Ca2+ is required to complete the later stages of apoptosis in DO11.10 cells, and its removal restores normal transport of PS, suggesting that down-regulation of the aminophospholipid translocase and up-regulation of the scramblase are not effected by irreversible protease cleavage.  (+info)

A novel beta 1 integrin-dependent mechanism of leukocyte adherence to apoptotic cells. (20/2206)

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.  (+info)

Endostatin induces endothelial cell apoptosis. (21/2206)

Endostatin, a carboxyl-terminal fragment of collagen XVIII, has been shown to regress tumors in mice. In this study, we have analyzed the mechanism of endostatin action on endothelial cells and nonendothelial cells. Endostatin treatment of cow pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay. Moreover, addition of endostatin led to a marked reduction of the Bcl-2 and Bcl-XL anti-apoptotic protein, whereas Bax protein levels were unaffected. These effects were not seen in several nonendothelial cells. Collectively, these findings provide important mechanistic insight into endostatin action.  (+info)

Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL. (22/2206)

TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) is a molecule that displays potent antitumor activity against selected targets. The results presented here demonstrate that human monocytes rapidly express TRAIL, but not Fas ligand or TNF, after activation with interferon (IFN)-gamma or -alpha and acquire the ability to kill tumor cells. Monocyte-mediated tumor cell apoptosis was TRAIL specific, as it could be inhibited with soluble TRAIL receptor. Moreover, IFN stimulation caused a concomitant loss of TRAIL receptor 2 expression, which coincides with monocyte acquisition of resistance to TRAIL-mediated apoptosis. These results define a novel mechanism of monocyte-induced cell cytotoxicity that requires TRAIL, and suggest that TRAIL is a key effector molecule in antitumor activity in vivo.  (+info)

On the mechanism of hepatic transendothelial passage of large liposomes. (23/2206)

Liposomes of 400 nm in diameter can cross the 100-nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid-type but not solid-type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS-containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.  (+info)

The yeast multidrug resistance pump, Pdr5p, confers reduced drug resistance in erg mutants of Saccharomyces cerevisiae. (24/2206)

Mutants of Saccharomyces cerevisiae bearing lesions in the ergosterol biosynthetic pathway exhibit a pleiotropic drug-sensitive phenotype. This has been reported to result from an increased permeability of the membranes of the mutant strains to different drugs. As disruption of the yeast multidrug resistance protein, Pdr5p, results in a similar pleiotropic drug-sensitive phenotype, the possibility that Pdr5p may be functioning with a reduced efficiency in these altered sterol backgrounds was examined. To do this, the function of Pdr5p in isogenic strains of S. cerevisiae that have disruptions in the late stages of the ergosterol biosynthesis pathway (ERG6, ERG2, ERG3, ERG4) was studied. A reduced ability of Pdr5p to confer resistance to different drugs in these strains was observed, which did not appear to be dependent solely on the permeability of the membrane towards the drug. A simultaneous examination was made of how the lipid composition might be altering the efficiency of Pdr5p by similar studies in strains lacking phosphatidylserine synthase (encoded by CHO1). The results indicated that the drug sensitivity of the erg strains is, to a significant extent, a result of the reduced efficiency of the Pdr5p efflux pump, and that the membrane environment plays an important role in determining the drug resistance conferred by Pdr5p.  (+info)