Thiol oxidation enforces phosphatidylserine externalization in apoptosis-sensitive and -resistant cells through a deltapsim/cytochrome C release-dependent mechanism

Previous studies have shown that unlike most apoptotic cells, Raji cells do not externalize phosphatidylserine (PS) upon apoptosis. Here we show that Raji cells are resistant to intrinsic apoptogenic agents, but sensitive to extrinsically triggered Fas-induced apoptosis. Treatment of intrinsic apoptosis-competent Jurkat cells with vitamin E implicated reactive oxygen species in intrinsic apoptosis because, like Raji cells, they became resistant to actinomycin D- but not Fas-triggered apoptosis. Oxidation of sulfhydryls in both cell types with N-ethylmaleimide resulted in rapid disruption of the mitochondrial membrane potential, release of cytochrome c from the mitochondria to the cytoplasm, and externalization of PS by a mechanism that was not inhibited by the pan caspase inhibiter zVAD-fmk. These results suggest that although cell death and PS externalization are both cytochrome c-dependent, they are distinct and separable processes.     

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.     

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.     

Apoptotic cells as sources for biologically active oxidized phospholipids

Acute inflammation is characterized by an accumulation of polymorphonuclear cells (PMNs), generation of reactive oxygen species, subsequent apoptosis of PMNs, and finally phagocytosis of apoptotic cells by macrophages. Recently, it has been demonstrated that during apoptosis oxidation of membrane phospholipids, especially phosphatidylserine, occurs. Moreover, we have shown that membrane vesicles released from apoptotic cells contain biologically active oxidized phospholipids. The involvement of oxidized phospholipids in the development of atherosclerosis, which is described as a chronic inflammatory disease, is increasingly recognized. These oxidized phospholipids were shown to induce several proinflammatory genes, such as monocyte chemoattractant protein 1 or interleukin-8, and it is hypothesized that lipid oxidation products also play a role in other chronic inflammatory disorders. On the other hand, oxidized phospholipids were shown to exert antiendotoxin effects by inhibiting lipopolysaccharide-induced signaling, representing a possible feedback loop during gram-negative infection. Additionally, it has been described that oxidized phospholipids are capable of inducing genes such as heme oxygenase-1 that are important for the resolution of acute inflammation. Moreover, oxidized phospholipids serve as recognition signals on apoptotic cells facilitating phagocytosis. In this review, we discuss the hypothesis that oxidized phospholipids generated in apoptotic cells (a) propagate chronic inflammation and (b) contribute to the resolution of acute inflammation.     

Apoptotic killing and phagocytosis of host cells by the parasite Entamoeba histolytica

The ability of Entamoeba histolytica to kill and phagocytose host cells correlates with parasite virulence. This study addressed the role of apoptotic cell killing and host cell phosphatidylserine exposure in the subsequent phagocytosis of Jurkat T cells by E. histolytica. Ingested host cells were apoptotic, as evidenced by the activation of caspase 3 in 88% +/- 3% (mean and standard deviation [SD] of the mean) of Jurkat cells engulfed by E. histolytica; ingested cells without detectable active caspase 3 were already disrupted and partially digested. That apoptotic cell killing preceded phagocytosis was supported by the demonstration that a higher percentage of amebae ingested apoptotic cells than ingested healthy cells (62% +/- 7% versus 30% +/- 9%, respectively [mean and SD]) (P = 0.008). E. histolytica also ingested apoptotic Jurkat cells more rapidly than necrotic control cells (8.5% +/- 0.4% versus 3.5% +/- 0.7%, respectively [mean and SD]) (P < 0.001). The inhibition of amebic cytotoxicity with D-galactose (which blocks the amebic Gal/GalNAc lectin) blocked the phagocytosis of healthy cells by greater than 80%, providing further evidence that apoptosis preceded engulfment. In contrast, D-galactose blocked the phagocytosis of already apoptotic cells by only 40%, implicating an additional host ligand (besides D-galactose) in amebic engulfment of apoptotic cells. The most characteristic surface change on apoptotic cells is phosphatidylserine exposure. Consistent with a role for host cell phosphatidylserine exposure in amebic ingestion of killed cells, Jurkat cell phosphatidylserine was exposed during incubation with E. histolytica (27% +/- 1% [mean and SD] specific increase at 30 min) (the P value versus the control was 0.0003). Approximately 50% more amebae ingested viable Jurkat cells expressing phosphatidylserine on the outer leaflet of the plasma membrane than ingested control cells (30.3% +/- 2.2% versus 19.8% +/- 1.9%, respectively [mean and SD]) (P = 0.003). By analogy with phagocytic clearance during apoptosis in metazoans, amebic apoptotic host cell killing followed by phagocytosis may limit inflammation and enable amebae to evade the host immune response.     

Progesterone-modulated phosphatidylserine externalization in apoptosis and activation of Jurkat cells

PROBLEM: During pregnancy the elevated levels of progesterone (Pg) have immunomodulating effects. It is important to follow-up Pg effects on basic biological processes at cell level as apoptosis and activation which was the aim of this study. METHODS OF STUDY: Jurkat cells cultured in the presence or absence of Pg were used as a model system. Apoptosis was induced by H(2)O(2) and activation by phorbol myriastate acetate. The induced changes in the phosphatidylserine (PS) externalization and cell surface CD69 expression were followed by fluorescence-activated cell sorter and immunofluorescence. RESULTS: After the induction of apoptosis PS externalizes in 52.3% of Jurkat cells. Cells cultured with Pg show tendency to a decrease of PS positive cells (42%). The opposite effect is observed in activated cells--PS externalization increase from 33.8% of control cells to 40.1% of Pg-treated cells. CONCLUSIONS: These findings would suggest that by increasing activation and decreasing apoptosis Pg could regulate local immune system during pregnancy.     

Disruption of the cytoskeleton after apoptosis induction with autoantibodies

F-actin cleavage was studied in PBMC after treatment with anti-dsDNA antibodies. Significant changes in F-actin disruption detected by decrease of FITC-phalloidin staining occurred after apoptosis induction with anti-dsDNA antibodies (p < 0.006). Despite of similar F-actin disruption, the switch of phosphatidylserine (PS) to the outer leaflet of the cell membrane as detected by annexin V binding was lower after anti-dsDNA antibody than without antibody treatment (58.4 +/- 11.0% vs. 81.9 +/- 7.7%). F-actin disruption was accompanied by activation of caspase 3 within the cytoplasm (r = -0.92599; p < 8.87446 x 10-(10)) under both conditions with and without autoantibodies. These findings indicate that anti-dsDNA antibody-induced apoptosis is more marked within the cell than upon the cell surface. The diminished externalization of PS might result in a decreased phagocytosis. Thereby, the reduced clearance of apoptotic cells could induce autoantibody production possibly against epitopes which arise due to the apoptotic disruption of cells.     

Phosphatidylserine-containing liposomes: potential pharmacological interventions against inflammatory and immune diseases through the production of prostaglandin E(2) after uptake by myeloid derived phagocytes

Phosphatidylserine (PS), which is normally located on the inner leaflet of the plasma membrane, translocates to the outer leaflet at the early stage of apoptosis. The PS externalization provides a signal for phagocytes to initiate uptake of apoptotic cells. After phagocytosis of apoptotic cells, phagocytes induce the secretion of anti-inflammatory mediators including prostaglandin E₂ (PGE₂). PS-containing liposomes (PSLs) can mimic the effects of apoptotic cells on phagocytes to induce the secretion of PGE₂. PSLs induce the PGE₂ secretion from microglia without induction of either cyclooxygenase (COX)-2 or microsomal prostaglandin E synthase (mPGES)-1. PSLs are found to rather utilize COX-1/mPGES-2 system to produce PGE₂ secretion and then shift microglia and macrophages from pro- to anti-inflammatory phenotype by an autocrine action of PGE₂. Moreover, PSLs inhibit the maturation of dendritic cells and osteoclast precursors. Therefore, PSLs will be potential pharmacological interventions for inflammatory and immune diseases through feedback mechanism utilizing PGE₂.     

小胶质细胞体外吞噬模型的建立及相关炎症因子的研究

目的: 建立以细胞凋亡信号－磷脂酰丝氨酸(PS)介导的小胶质细胞体外吞噬模型,研究小胶质细胞发挥吞噬功能时炎症因子的表达变化。方法: 用N-乙基马来酰胺(NEM)预处理红细胞,随后整合氧化PS,制备含氧化PS信号的红细胞凋亡模型,并测定小胶质细胞对整合氧化PS信号红细胞吞噬率的变化。同时, real-time PCR检测小胶质细胞中炎症因子白细胞介素1β(IL-1β)和肿瘤坏死因子(TNF-α)的表达情况,研究小胶质细胞吞噬功能与炎症功能之间的关系。结果: 小胶质细胞对整合氧化PS红细胞的吞噬率显著高于对照组(P<0.01),同时炎症因子IL-1β和TNF-α的mRNA表达水平明显减少。结论: 成功制备小胶质细胞体外吞噬凋亡细胞模型,小胶质细胞的吞噬作用可能是自身炎症因子IL-1β和TNF-α在转录水平上降低的诱因。     

Oxidized Phosphatidylserine: Production and Bioactivities

Recent development of analytical methods for lipid hydroperoxides and preparation of highly pure lipid hydroperoxides have revealed the important new pathophysiological roles of oxidized phospholipids. Generation of reactive oxygen species and subsequent oxidative stress leads to random oxidation of membrane phospholipids. However, recent studies have reported that anionic phospholipid molecules such as phosphatidylserine (PS) and cardiolipin are preferentially oxidized during apoptosis, resulting in efficient apoptosis execution and apoptotic cell clearance by phagocytes. This review is exclusively focused on selective production of oxidized PS (oxPS) during apoptosis as well as the novel roles of oxPS under pathophysiological conditions.     

Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal--implications for antiphospholipid syndrome and systemic lupus erythematosus

In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.     

Recognition of apoptotic cells by human peripheral blood monocytes does not alter their ability to phagocytize and kill Staphylococcus aureus

INTRODUCTION: During acute inflammation, leukocyte infiltration is mostly neutrophilic, but later monocytes prevail. The majority of inflammatory cells, particularly neutrophilic polymorphonuclear leukocytes (PMNs), become apoptotic at later stages of inflammation and are phagocytosed by neighboring cells, mostly by macrophages. Recently, it has been found that human peripheral blood monocytes also recognize apoptotic cells, which primes them to increased production of interleukin (IL)-10--a cytokine known to reduce phagocytes' ability to engulf and kill pathogens. Based on the above, we studied monocytes' ability to phagocytose and kill Staphylococcus aureus while in contact with apoptotic cells. MATERIALS AND METHODS: Monocytes isolated by elutriation were co-cultured with apoptotic PMNs or Jurkat cells and exposed to viable, human serum-opsonized S. aureus. To induce apoptosis PMNs were cultured overnight while Jurkat cells were UV-treated. Apoptosis, phagocytosis of bacteria and intracellular superoxide production were measured by flow cytometry. Production of reactive oxygen species was also followed by measurement of chemiluminescence. The bactericidal effect was determined by standard colony forming units method. RESULTS: Data presented show that contact of monocytes with apoptotic neutrophils and Jurkat cells had no influence on monocyte phagocytosis of S. aureus, the generation of reactive oxygen species, or the killing of bacteria. CONCLUSION: The data obtained suggest that monocytes attracted to the inflammatory site are not deficient in their ability to cope with pathogens after contact with apoptotic cells despite increased production of IL-10.     

Entamoeba histolytica cell surface calreticulin binds human c1q and functions in amebic phagocytosis of host cells

Phagocytosis of host cells is characteristic of tissue invasion by the intestinal ameba Entamoeba histolytica, which causes amebic dysentery and liver abscesses. Entamoeba histolytica induces host cell apoptosis and uses ligands, including C1q, on apoptotic cells to engulf them. Two mass spectrometry analyses identified calreticulin in amebic phagosome preparations, and, in addition to its function as an endoplasmic reticulum chaperone, calreticulin is believed to be the macrophage receptor for C1q. The purpose of this study was to determine if calreticulin functions as an E. histolytica C1q receptor during phagocytosis of host cells. Calreticulin was localized to the surface of E. histolytica during interaction with both Jurkat lymphocytes and erythrocytes and was present in over 75% of phagocytic cups during amebic erythrophagocytosis. Presence of calreticulin on the cell surface was further demonstrated using a method that selectively biotinylated cell surface proteins and by flow cytometry using trophozoites overexpressing epitope-tagged calreticulin. Regulated overexpression of calreticulin increased E. histolytica's ability to phagocytose apoptotic lymphocytes and calcium ionophore-treated erythrocytes but had no effect on amebic adherence to or destruction of cell monolayers or surface expression of the GalNAc lectin and serine-rich E. histolytica protein (SREHP) receptors. Finally, E. histolytica calreticulin bound specifically to apoptotic lymphocytes and to human C1q. Collectively, these data implicate cell surface calreticulin as a receptor for C1q during E. histolytica phagocytosis of host cells.     

Levels of complement in sera from inactive SLE patients, although decreased, do not influence in vitro uptake of apoptotic cells

BACKGROUND: Accumulation of apoptotic cells is considered relevant in the pathogenesis of systemic lupus erythematosus (SLE). Complement factors facilitate the clearance of apoptotic cells and, when decreased, might result in an increased amount of apoptotic cells found in SLE patients. OBJECTIVE: To determine the influence of complement profiles from inactive SLE patients on the in vitro phagocytosis of apoptotic cells. METHODS: Consecutive SLE patients (n=98) with inactive disease (SLEDAI < or =4) and 20 healthy controls (HC) were included. Levels of CH50, C3, C4, C1q, and C1r were measured. Human peripheral blood monocytes were isolated from healthy controls and cultured for 7 days to obtain monocyte-derived macrophages (MDM). Jurkat cells were irradiated with UVB to induce apoptosis. Phagocytosis was tested by incubation of MDM with apoptotic cells in the presence of serum and quantified as phagocytosis index (number of Jurkat cells internalized by 100 macrophages). Serum from 20 patients with CH50<65%, 20 patients with CH50 > or =65%, and 20 HC were used in this assay. RESULTS: All HC and 37% of patients had normal complement levels. CH50 level was decreased in 21% of patients, C3 in 52%, C4 in 29%, C1q in 2% and C1r in 44% of patients. Between patients and HC, differences in level of CH50, C3 and C4 were statistically significant. No difference in phagocytosis index between HC and patients, irrespective of their CH50 level, was detected. No correlation was found between the respective complement levels and phagocytosis index. CONCLUSION: In most SLE patients with inactive disease, levels of one or more complement components are decreased. However, decreased levels of complement do not result in a significantly reduced in vitro uptake of apoptotic Jurkat cells by MDM.     

Mechanisms Underlying Production And Externalization of Oxidized Phosphatidylserine in Apoptosis: Involvement of Mitochondria

The present study was performed by using selective inhibitors of caspase-8 and caspase-3 functioning upstream and downstream from mitochondria, respectively to determine whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, release of cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced mitochondrial cyt c release, the amount of PSox not only within but also on the surface of Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on the surface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation and apoptosis. Thus, these results strongly suggest that mitochondria play an important role in production of PSox and subsequent its externalization during apoptosis.     

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.     

TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells

The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.     

Possible contribution of apoptosis-inducing factor (AIF) and reactive oxygen species (ROS) to UVB-induced caspase-independent cell death in the T cell line Jurkat

We analyzed the mechanism of UVB-induced cell death using the Jurkat T cell line. Apoptosis was assessed by measuring phosphatidylserine (PS) externalization, caspase activity, the decrease in mitochondrial membrane potential (Delta Psi m), nucleosomal DNA fragmentation, and morphological changes such as chromatin condensation. The mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF) was evaluated by confocal laser microscopy. The cell death pattern of UVB-irradiated cells was similar to the Fas-induced cell death pattern. However, zVAD-fmk inhibited the nucleosomal fragmentation of DNA but not the externalization of PS, decrease in Delta Psi m, or mitochondrio-nuclear translocation of AIF. N-acetyl L-cysteine significantly inhibited the translocation of AIF induced by UVB. These results suggested that caspase-dependent and -independent pathways were involved in UVB-induced cell death in Jurkat cells, and the mitochondrio-nuclear translocation of AIF was associated with the latter pathway. In addition, reactive oxygen species generated by UVB might be involved in inducing the mitochondrio-nuclear translocation of AIF.     

Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm quality

BACKGROUND: The aim of the present study was to define the effect of apoptosis on sperm quality and function. METHODS: The apoptotic features in sperm were assessed in 60 subfertile subjects, using Annexin-V staining for phosphatidylserine (PS) externalization and Tdt-mediated dUTP nick end labelling (TUNEL) assay for DNA fragmentation. RESULTS: On average, about 45% of the sperm were found to be apoptotic based on the results from Annexin-V staining, including both early (Annexin-V-positive, PI-negative) and late apoptosis (Annexin-V-positive, PI-positive). TUNEL-positive cells (median value 15%) significantly correlated to late apoptosis but not early apoptosis, indicating that DNA fragmentation only occurs at the later stage of sperm apoptosis. TUNEL-positive and late apoptotic cells (Annexin-V-positive, PI-positive) were found to be inversely correlated to sperm motility and vitality, and positively to abnormal sperm morphology. On the other hand, it is surprising to note that the apoptotic alterations in sperm positively correlated to sperm concentration or total sperm counts. CONCLUSIONS: Overall results from this study support the abortive apoptosis theory; apoptosis in mature sperm is initiated during spermatogenesis, after which some cells earmarked for elimination via apoptosis may escape the removal mechanism and contribute to poor sperm quality.