Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.     

Fas activation reduces neutrophil adhesion to endothelial cells

Polymorphonuclear neutrophils (PMN) express apoptotic markers and lose effector functions including adhesion, chemotaxis, and phagocytosis when cultured overnight. Although the loss of function correlates with apoptosis, it is not clear if functions are lost before an early marker of apoptosis, the display of phosphatidylserine (PS), targets PMN for removal by phagocytic cells. To address this question, freshly isolated PMN were treated with Fas-activating antibodies to induce apoptosis rapidly. Early markers of apoptosis and PMA-stimulated adhesion to endothelial cells were measured. After 1 h of Fas exposure, only 16% PMN had externalized PS. In contrast, Fas activation reduced PMA-stimulated adhesion between 68 and 27% depending on PMA concentration. The loss of adhesion was accompanied by a reduction in beta2 integrin expression and receptor clustering. These results indicate that the Fas-induced loss of adhesion may precede PS externalization and could limit participation in the inflammatory response before PS externalization targets PMN for removal.     

The mode of action of the calcium ionophore A23187 on T cell proliferation. I. The ionophore does not replace lymphokines but acts via induction of IL-2 production on IL-2 responsive cells

The two compounds, the calcium ionophore A23187 and phorbol myristate acetate (PMA), which are not mitogenic for mouse thymocytes, induce proliferation when added in combination to thymocyte cultures. Short exposure to PMA renders the cells responsive to IL-2, added exogeneously. The cells rendered IL-2-responsive by PMA are enriched in the more mature peanut agglutinin (PNA)-negative population. The ionophore does not render PNA-negative thymocytes IL-2-responsive, but induces proliferation of PMA-pulsed PNA-negative thymocytes. PMA-pulsed PNA-negative thymocytes proliferating in response to either exogeneous IL-2 or ionophore express IL-2 receptors. However, the ionophore does not mimic IL-2 action but acts indirectly by induction of both IL-2 production and of IL-2 receptor expression via IL-2. This view is based on the following findings: 1) IL-2 could be detected in supernatants derived from PMA-preactivated thymocytes incubated with the ionophore; 2) The IL-2-induced proliferation, as well as the ionophore-induced proliferation, was specifically blocked by a monoclonal anti-IL-2-receptor antibody; 3) The proliferation induced by exogeneous IL-2, as well as that induced by the ionophore, could be specifically inhibited by metabolically blocked T lymphoblasts carrying IL-2 receptors competing with the responder cells for the available IL-2 added or produced in the system.     

Reversible phosphatidylserine expression on blood granulocytes related to membrane perturbation but not DNA strand breaks

Exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane is recognized as an early indicator of programmed cell death (apoptosis) in plant and mammalian cells. Currently, there is no literature describing that PS expression on the surface of white blood cells is reversible. We found that a hypotonic 0.2% NaCl or NH(4)Cl lysing solution used to separate white blood cells from red blood cells induced a reversible PS expression on the cell surface of granulocytes and monocytes but not lymphocytes. This reversible PS expression was associated with change of plasma membrane potential but not degranulation-associated membrane mobilization or DNA fragmentation. In contrast, TNF-alpha induced an irreversible PS expression, associated with apoptotic DNA fragmentation shown on gel electrophoresis. The fact that hypotonic shock induced a reversible PS expression on granulocytes, and TNF-alpha induced an irreversible PS expression associated with apoptotic DNA fragmentation indicate the new insight that expression of PS on the outer cell surface does not always represent cell apoptosis. Also, the reversible PS expression was associated with altered plasma-membrane potential but not DNA strand breaks, indicating that early PS expression may be related to the membrane perturbation but not directly related to DNA fragmentation in certain types of cells.     

SH-SY5Y细胞凋亡过程中Cpne5蛋白表达的变化

目的探讨Cpne5蛋白在SH-SY5Y细胞凋亡中表达量的变化。方法显微镜观察细胞形态及流式细胞术检测凋亡率,鉴定A23187诱导SH-SY5Y细胞凋亡及血清剥夺诱导凋亡的模型;免疫印迹法检测两种凋亡过程中Cpne5蛋白表达量的变化。结果 10μmol/L A23187诱导SH-SY5Y细胞24h,细胞出现皱缩、凋亡率增加。A23187诱导0.5h,Cpne5蛋白表达量即出现明显的上升,持续至12h;在24h时Cpne5蛋白表达量回落至诱导前水平。血清剥夺72h的SH-SY5Y细胞24份,凋亡率为23.2±2.1%,与对照组1.1±0.1%比,差异有统计学意义（P〈0.01）。免疫印迹结果表明,SH-SY5Y细胞分别经血清剥夺1h、6h、12h、24h、72h对Cpne5蛋白表达量无明显变化。结论 A23187诱导SH-SY5Y细胞凋亡早期Cpne5蛋白表达量有显著变化;提示Cpne5基因可能参与凋亡调控的钙信号通路。     

Human cytomegalovirus induces apoptosis in the hematopoietic cell line MO7e

Several studies have shown that human cytomegalovirus (HCMV) induces growth suppression of hematopoietic progenitors. In vitro studies have demonstrated that the HCMV-induced suppression is independent of viral protein production. Previous studies have indicated a link between HCMV infection and apoptosis in human cells. The purpose of our study was to investigate whether the observed inhibitory effect of HCMV on the human myeloid progenitors could be connected to the induction of apoptosis. The growth and cell death of the hematopoietic cell line MO7e was investigated following infection with HCMV virions and dense bodies. Both virions and dense bodies inhibited the growth of MO7e cells, and induced cell death measured by trypan blue staining. In addition, both HCMV virions and dense bodies caused an increased amount of apoptosis-characteristic DNA fragmentation in the MO7e cells compared to mock-treated cells. The HCMV virions were also able to induce an increased expression of phosphatidylserine on the cell surface, which is an early event in the initiation of apoptosis in most cell types. In conclusion, HCMV and HCMV dense bodies are able to induce apoptosis in the myeloid progenitor cell line MO7e.     

The plasma membrane is the site of selective phosphatidylserine oxidation during apoptosis: role of cytochrome C

Phosphatidylserine (PS) externalization, a functional end point of apoptosis that triggers phagocytic recognition of dying cells, may be modulated by oxidative stress in biological membranes. We previously observed selective oxidation of PS during apoptosis, but the intracellular location and molecular mechanisms responsible for PS oxidation remain to be described. Peroxidation in individual classes of cellular phospholipids was monitored in whole cells and various subcellular fractions obtained from HL-60 cells undergoing apoptosis in response to tert-butyl hydroperoxide (t-BuOOH) after metabolic acylation of phospholipids with the oxidation-sensitive fluorescent fatty acid, cis-parinaric acid. Nonrandom selective oxidation of PS was observed in whole cells, as well as in plasma membrane. PS in mitochondria appeared selectively resistant to oxidation during apoptosis. All phospholipids in nuclear membranes appeared resistant to oxidation after t-BuOOH treatment. Selective PS oxidation was accompanied by cytochrome c release and PS externalization. PS oxidation and externalization were followed by caspase activation and other end points of apoptosis. HL-60 cells "loaded" with exogenous cytochrome c by mild sonication showed selective oxidation of PS in both the absence and presence of t-BuOOH. Cytochrome c/hydrogen peroxide could effectively oxidize purified PS but not phosphatidylcholine in a cell-free model system. Selective plasma membrane-based PS oxidation and subsequent externalization during oxidant-induced apoptosis may be mediated through the redox activity of cytochrome c.     

Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis

In glucocorticoid-treated rat thymocytes and the murine lymphoid cell lines L5178 and S49 the morphology of apoptosis is associated with chromatin cleavage. The cleavage is at internucleosomal sites, apparently through activation of an endogenous endonuclease. In variants of the cell lines selected for resistance to glucocorticoid, neither apoptosis nor chromatin cleavage were observed after steroid treatment, and steroid receptors were undetectable. In thymocytes, both the morphological changes of apoptosis and chromatin cleavage were inhibited by cycloheximide and actinomycin D. The calcium-magnesium ionophore A23187 induced apoptosis and chromatin cleavage in thymocytes, and these effects were also inhibited by cycloheximide. The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments. They also provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.     

Calcium ionophore-induced apoptosis of human B cells is preceded by the induced expression of early response genes

We have investigated the effects of calcium ionophore on apoptosis of Ramos human B cells. Our results show that the calcium ionophore A23187 at denned concentrations leads to apoptosis of Ramos cells. The majority of cells (> 90%) undergo apoptosis in response to ionophore. The response is rapid and nuclear condensation and DNA degradation can be detected within 2 h after addition of ionophore. In attempts to define the changes in gene expression preceding apoptosis, we investigated the expression of a panel of early response genes in these cells after ionophore addition. We show that calcium ionophore-induced apoptosis of Ramos cells is preceded by the induced expression of a number of early response genes. These results are consistent with calcium ionophore initiating changes in gene expression which may be important in signaling these cells to undergo apoptosis.     

Key role of mitochondria in apoptosis of lymphocytes

Changes in the mitochondrial potential, expression of phosphatidylserine, parameters of direct and lateral light scattering, and DNA fragmentation during spontaneous and induced apoptosis in peripheral blood lymphocytes were studied by flow cytofluorometry. Dexamethasone and Ca²⁺ ionophore A23187 served as inductors of apoptosis. A decrease in the mitochondrial potential is an early sign of spontaneous and induced apoptosis. Phosphatidylserine expression on the outer plasma membrane occurred later and inversely depended on the mitochondrial potential. Our results indicate that the involvement of mitochondria in spontaneous and induced apoptosis accompanied by a decrease in the mitochondrial potential is an early and key event of programmed lymphocyte death. The decrease in the mitochondrial potential of lymphocytes induced degradation of their nuclei (DNA fragmentation) and promoted elimination of apoptotic cells (phosphatidylserine expression).     

CD3-mediated apoptosis of human medullary thymocytes and activated peripheral T cells: Respective roles of interleukin-1, interleukin-2, interferon-γ and accessory cells

Clonal deletion represents an important mechanism for the establishment of tolerance, by the elimination of autoreactive T cells. Deletion is accomplished by programmed cell death, termed apoptosis, induced by mobilization of the T cell receptor (TCR) on both thymocytes and mature T cells. The mechanism which drives T cells towards cell death or cell proliferation after TCR mobilization remains unclear. We show here that the mobilization of the CD3/TCR complex of both CD4⁺ and CD8⁺ single-positive medullary human thymocytes and human mature activated T cells, in the absence of accessory cells, leads to an activation-induced cell death process by apoptosis. In both cases, apoptosis was associated with interferon (IFN)-γ gene expression and secretion in the absence of interleukin (IL)-2 gene expression; and the addition of anti-IFN-γ antibody prevented cell death. Apoptosis could also be prevented by cyclosporin A (CsA) treatment and could be re-induced by the addition of IFN-γ to CsA-treated cells. Addition of IL-2 had two different effects, it prevented apoptosis and also allowed proliferation in response to CD3 monoclonal antibody. Addition of IL-1, which induces IL-2 gene expression and secretion or addition of accessory cells, had the same preventive effect. These results suggest that the uncoupling of IFN-γ and IL-2 gene expression following CD3/TCR mobilization initiates apoptosis of human T cells at several different stages during development and activation. We propose that co-signals provided by accessory cells allow a coupling of IL-2 gene and IFN-γ gene expression, and that an essential role for IL-2 secretion in T cell activation involves the inhibition of a death program induced by IFN-γ secretion.     

Vitamin E inhibits anti-Fas-induced phosphatidylserine oxidation but does not affect its externalization during apoptosis in Jurkat T cells and their phagocytosis by J774A.1 macrophages

Apoptosis and phagocytosis of apoptotic cells provide for effective and harmless clearance of unwanted or damaged cells in the body. Preferential oxidation of one particular class of phospholipids, phosphatidylserine (PS), is a typical trait of both oxidant- and nonoxidant-induced apoptosis. PS oxidation is likely to play an important role in phagocytosis either by affecting PS externalization acting as an "eat me" signal or by more effective recognition of apoptotic cells by macrophage receptors. This implies that antioxidants effective in inhibiting PS oxidation may affect PS externalization and/or effective removal of apoptotic cells. Therefore, it is essential to determine whether vitamin E, the major lipid-soluble antioxidant of membranes, inhibits PS oxidation, and hence blocks apoptosis/phagocytosis. To test this, we studied the effects of vitamin E on PS oxidation and signaling using a model of anti-Fas-triggered apoptosis in Jurkat T cells. We found that incubation of cells with vitamin E (0.25-50 micro M) resulted in its integration into cells to reach physiologically relevant concentrations. Using labeling of cell phospholipids with oxidation-sensitive and fluorescent cis-parinaric acid (PnA), we found that anti-Fas exposure caused significant and selective oxidation of PnA-PS in Jurkat T cells (22 +/- 2.1% of its content in nonexposed cells). Vitamin E protected PnA-PS against oxidation in a concentration-dependent way such that at 25 micro M and 50 micro M, a complete inhibition of anti-Fas-induced PS oxidation was achieved. At all concentrations used, vitamin E had no effect on either biomarkers of anti-Fas-induced apoptosis (PS externalization, nuclear fragmentation) or phagocytosis of anti-Fas-induced apoptotic cells by J774A.1 macrophages. We conclude that vitamin E does not significantly interfere with extrinsic (death receptor-triggered) pathways of apoptosis and does not affect phagocytosis of anti-Fas-triggered apoptotic cells.     

TCR抗体诱导不成熟胸腺细胞亚群凋亡的敏感性研究

为比较小鼠不同胸腺细胞亚群对抗TCR抗体诱导凋亡的敏感性，用体外抗TCR抗体刺激分离胸腺细胞．BALB／c小鼠体内注射抗TCR抗体，FACS检测胸腺细胞。结果显示，CD4^ CD8^ DP胸腺细胞和CD4^-CD8^ CD3^-TCR-细胞对抗TCR抗体诱导的凋亡敏感，但CD4^-CD8^-CD3^-TCR-胸腺前体细胞自发凋亡率低，且抗TCR抗体诱导的凋亡．表明胸腺细胞对凋亡的敏感点产生于CD4^-CD8^ CD3^-TCR-细胞表达后，胸腺细胞的凋亡敏感性受发育调节。     

CD28协同刺激分子对T细胞受体介导的胸腺细胞亚群凋亡的影响

目的：分析anti-TCRαβmAb anti-CD28mAb诱导小鼠胸腺淋巴细胞不同亚群的凋亡及凋亡程度，分析CD28协同刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响。方法：新鲜分离胸腺细胞，加入anti-TCRαβmAb-anti-TCRαβmAb anti-CD28mAb等培养20h，进行多重染色，流式细胞仪分析。结果：与胸腺细胞自发凋亡的结果相比较；（1）双信号刺激可明显增加胸腺细胞凋亡的数目，尤其是CD4^ CD8^ 胸腺细胞的凋亡数目。（2）凋亡的CD4^ CD8^ 亚群，CD4^ CD8^-亚群细胞表面CD28的表达均增多。结论：CD28共刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响与细胞的成熟程度有关，CD28共刺激分子能明显增强不成熟皮质胸腺细胞的凋亡。     

Effects of the thymic microenvironment on the response of thymocytes to stimulation.

We show that, in vitro, the response of thymocytes to certain stimuli, and their survival largely depend on the nature of the culture environment, i.e. whether thymocytes are stimulated within intact thymus lobes or in cell suspension. Exposure of isolated thymocytes to 12-O-tetra-decanoylphorbol 13-acetate (TPA)+ionomycin rapidly abolishes the expression of recombination-activating gene-1 (RAG-1) mRNA (3 h), down-regulates CD4 surface antigen expression (3 h), and enhances apoptosis (24 h). On the other hand, when thymocytes are cultured in intact lobes, TPA plus ionomycin down-regulate rather than abolish RAG-1 mRNA expression (3 h), have little effect on CD4 expression even following 24-h exposure, and only marginally induce apoptosis (24 h). Differences between the culture systems are less pronounced in response to anti-CD3 antibodies. Therefore, it appears that removing thymocytes from their thymic microenvironment makes the cells more susceptible to certain stimuli, possibly by altering their physiological status. In addition, it has been suggested that termination of RAG-1 expression can be linked to thymocyte selection processes. We found that the down-regulation of RAG-1 expression was not dependent on the induction of apoptosis, supporting a proposed link with positive selection.     

Tetrachlorodibenzo-p-Dioxin (TCDD) Inhibits Differentiation and Increases Apoptotic Cell Death of Precursor T-Cells in the Fetal Mouse Thymus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy as well as alterations in thymocyte maturity in mice. Multiple mechanisms for thymic hypocellularity have been suggested, and include an increase in thymocyte apoptosis, a maturation arrest of thymocyte development, inhibited thymocyte proliferation, and a diminution of seeding of the thymus by the hematopoietic progenitors in the fetal liver or adult bone marrow. Fetal mice are highly sensitive to hypocellularity induction by TCDD when the chemical is administered during the window of thymic development, between days 10 and 18 of gestation. Treatment of pregnant C57Bl/6 mice in the present experiments with doses of 5 or 10 mu g/kg TCDD by oral gavage on gestation days 14 and 16 severely depressed day 18 thymic cellularity. Histopathologic evaluation of day 18 fetal thymi showed disruption of the normal organ architecture with loss of clear distinction between cortical and medullary regions after TCDD. A decrease in thymocyte density was noted in all regions, and was most dramatic in the cortical zones where pyknotic cells were increased by TCDD treatment. Using day 18 thymocyte suspensions and flow cytometry, the marker 7-AAD showed a decrease in viable thymocytes from TCDD-treated fetal mice, and a concomitant and dose-related increase of thymocytes in early apoptosis. Specifically, relative to control, thymocytes from the 5 and 10 mug/kg TCDD exposure groups displayed 1.9% and 5.3% respective increases in early apoptotic cells. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, the enhanced apoptosis occurred in the CD4(+)CD8(+) phenotype with no significant apoptosis seen in the CD4(-)CD8(-), CD4(+)CD8(-), or CD4(-)CD8(+) thymocytes. Given the rapid clearance of apoptotic cells from the thymus, these histopathologic and cytometric data suggest increased thymocyte apoptosis contributes to fetal thymic atrophy after TCDD exposure.     

Apoptosis of thymic lymphoma clones by thymic epithelial cells: a putative model for 'death by neglect'

We have previously described an in vitro system in which thymic epithelial cells induce apoptosis in CD4+ 8+ thymocytes or thymic lymphoma cells, in the absence of an exogenous antigen. A thymic epithelial cell line (TEC) recapitulated the response, by inducing apoptosis in CD4+ 8+ thymocytes of the thymic lymphoma clone, PD1.6. The present study pursues the involvement of the T-cell receptor (TcR) in the response of PD1.6 to TEC. TcR cross-linking did not cause apoptosis of PD1.6, although it induced tyrosine phosphorylation of p95vav. In contrast, TEC did not induce phosphorylation of p95vav but induced apoptosis of PD1.6 cells. These results suggest that TcR-evoked signals are not involved in TEC-induced apoptosis of PD1.6. Intracellular calcium chelation, using BAPTA-loaded PD1.6 cells, diminished TEC-induced apoptosis. Protein kinase C depletion in PD1.6 cells augmented their apoptotic response to TEC. Thus, the response of PD1.6 to TEC is calcium-dependent and inhibited by PKC. Likewise, the apoptotic response of PD1.6 to A23187 was abrogated by PKC activation. PD1.6 cells may represent an immature double positive thymocyte population, which does not undergo negative selection. The interaction of PD1.6 with TEC may thus serve as a model for the TcR-independent 'Death by Neglect', which takes place in the thymus during thymocyte development.     

Thymic epithelial cells induce Fas-independent activation apoptosis of thymocytes

Co-cultivation of human thymocytes with homologous thymic epithelial cells (TEC) resulted in apoptosis of thymocytes and increase of CD25 expression. TEC supernatant also induced these effects. Fraction of apoptotic cells was enriched by CD69+ cells but not by CD95+ cells. Thymocytes of mice MRL-lpr/lpr bearing mutant form of gene Fas were sensitive to apoptosis induction in co-culture with TEC in the same degree as thymocytes of mice bearing Fas gene of wild type. Apoptosis of murine thymocytes can be induced by co-cultivation with both murine and human TEC.     

Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ-irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non-thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti-CD2 and anti-LFA-1 antibodies. The immature CD3^−/+dull CD4⁺CD8⁺ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC-induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid-induced thymocyte apoptosis is p53-independent, glucocorticoids are conceivably not involved in TEC-induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.     

Macrophages engulfing apoptotic thymocytes produce retinoids to promote selection,differentiation, removal and replacement of double positive thymocytes

The thymus provides the microenvironment in which thymocytes develop into mature T-cells, and interactions with thymic stromal cells are thought to provide the necessary signals for thymocyte maturation. Recognition of self-MHC by T-cells is a basic requirement for mature T-cell functions, and those thymocytes that do not recognize or respond too strongly to the peptide-loaded self-MHC molecules found in the thymus undergo apoptosis. As a result, 95% of the thymocytes produced will die and be subsequently cleared by macrophages. This review describes a complex crosstalk between developing thymocytes and engulfing macrophages which is mediated by retinoids produced by engulfing macrophages. The interaction results in the harmonization of the rate of cell death of dying double positive cells with their clearance and replacement, and in promotion of the differentiation of the selected cells in the thymus.