Apoptosis is an active cell death that requires intact subcellular structures and synthesis of phylogenetically conserved proteins. It characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in < 2 hours. Apoptosis arises from the active initiation and propagation of a series of highly orchestrated specific biochemical events leading to the demise of the cell^ref. Functional consequences of this process are the elimination of specific cells within a population when they are damaged or no longer required for function. For example, apoptosis is an important determinant for tissue morphogenesis during development^ref and also plays a role in the elimination of immune effector cells during lymphocyte selection^ref and the resolution of inflammation and fibrosis^ref. Failure of efficient apoptosis can allow the progression of neoplastic disease, permitting the persistence and multiplication of cells suffering significant genotoxic damage.
Variants of apoptosis include :

• lipo-apoptosis / lipoptosis : a balanced lipid metabolism is crucial for all cells. Disturbance of this homeostasis by nonphysiological intracellular accumulation of fatty acids can result in apoptosis. This was proven in animal studies and was correlated to some human diseases, like lipotoxic cardiomyopathy. Some metabolic mechanisms of lipo-apoptosis were described, and some causes were discussed, but reagents, which directly induce lipo-apoptosis, have thus far not been identified. The human monoclonal IgM antibody SAM-6 was isolated from a stomach cancer patient by using the conventional human hybridoma technology (trioma technique). The addition of SAM-6 to tumor cells leads to an increase in the intracellular accumulation of neutral lipids, followed by tumor cell apoptosis. The antibody SAM-6 does not react with noncancerous human epithelial and fibroblastic cells, because the M(r) 140000 membrane molecule, recognized by the antibody, is specifically expressed on human malignant cells. The antibody is coded by the germ-line genes IgHV3-30.3*01 and IgLV3-1*01 and is a component of the innate immunity to cancer^ref
• neosis : multinucleate/polyploid giant cells (MN/PGs) formed due to DNA damage in carcinogen-induced transformation of C3H10T1/2 cells are thought to die via mitotic catastrophe. Before they die, some MN/PGs undergo a novel type of cell division characterized by karyokinesis via nuclear budding followed by asymmetric, intracellular cytokinesis, producing several small mononuclear cells, termed the Raju cells, with extended mitotic life span (MLS). Mitotic derivatives of Raju cells give rise to transformed cell lines, inherit genomic instability, display a phenotype and transcriptome different from the neosis mother cell, and anchorage-independent growth. Neosis of MN/PGs also precedes spontaneous transformation of p53^-/- mouse cells. Rodent neotic clones, and primary and metastatic human tumor cells undergo spontaneous or induced secondary/tertiary neosis. Neosis seems to extend the MLS of cells under conditions of genetic duress not favoring mitosis. It precedes tumorigenesis, occurs several times during tumor progression, yielding tumor-initiating Raju cells and introducing tumor cell heterogeneity subject to natural selection during tumor progression. Anti-neotic agents (neosicides) could have therapeutic advantages^ref
• necroptosis : although research over the past decade^ref has shown that apoptosis is likely not the only type of programmed cell death, little is known about what other mechanisms may look like. The chemical necrostatin-1 (Nec-1) has no effect on apoptosis, but only on another programmed necrosis-like death, termed necroptosis. Although necroptosis shows some characteristics of autophagy, this is a downstream consequence of necroptotic signaling, not an upstream effector of it. There have been many hints that there are ways to kill cells other than the classical apoptotic pathway : the one thing that has been lacking so far has been a way to figure out what proteins are involved in these other pathways. A canonical apoptotic pathway is triggered when ligands bind to members of the Fas/TNFR family of death-domain receptors. This pathway sequentially turns on multiple caspases, which are cysteine proteases that effect apoptotic cell death. Some work has shown, however, that the Fas/TNFR family can induce cell death even when caspase signaling—and therefore apoptosis—is inhibited^ref. Also, cell death under these conditions usually looks more like necrosis than apoptosis. There are a number of forms of programmed cell death that have been identified that have more necrotic-like phenotypes. What's been missing is proof that a common pathway carries out nonapoptotic programmed cell death in the multiple types of cells in which it's been observed. When human cells were treated with TNF-a, a ligand for the Fas/TNFR family, along with a pan-caspase inhibitor, many cells died a necrosis-like death, as expected. Nec-1, however, that prevented these cells from dying : it inhibited TNFa-induced necrosis in all of them. This is the first direct evidence of a common, alternative form of programmed cell death. Since it's well known that ischemic brain injury, such as that seen in stroke, involves both apoptotic and nonapoptotic PCD^ref, injecting necrostatin-1 into mouse ventricles significantly reduced the volume of dead brain tissue after stroke-like injury. This protection suggests that necroptosis is involved in this form of pathologic cell death.

• the volume of the targeted cell decreases (cytosolic shrinkage) on the contrary of oncosis => condensation of the cell
• more marked chromatin margination
• nuclear condensation
• redistribuction of nucleus fragments to blebs on the apoptotic cell surface


• cytoplasmic blebbing
• swollen ER on occasion
• mitochondria normal or condensed
• transglutaminase creates nets and so apoptotic bodies can be phagocytated

• activation-induced cell death (AICD) : overexpression of receptors for pro-apoptotic ligands (e.g. in negative selection of T lymphocytes, infected or stressed cells, activated macrophages, activated T and B lymphocytes)
• hypothermy
• hyperthermy
• retinoic acid
• glucocorticoids
• 20-hydroxyecdisone
• T₃
Activation of the ERK pathway, which is downstream of TLR signalling, and activity of the myocyte-specific enhancer binding factor 2 (Mef2), which is upregulated by the pan-caspase inhibitor zVAD, are both required for the induction of NR4A1 / NUR77 transcription and for NUR77-mediated caspase-independent macrophage cell death.
• programmed cell death (PCD) : innate tendency in absence of external stimuli such as
• growth factors (e.g. neurons and interdigital membrane in embryogenesis and uterine mucosa during menstrual cycles)
• self-MHC molecules (e.g. in positive selection of T lymphocytes)
• ECM anchorage : ainokis / detachment-induced apoptosis is the programmed cell death occurring in most untransformed cell types (except for blood cells) when anchorage to ECM is removed. It is induced by tropomyosin-1, BAD overexpression and repressed by merosin.
• cell-autonomous cell death : viral infection or excessive DNA damage trigger apoptosis in the affected cells through PKR- and p53-dependent pathways, respectively.

• in the lung, as in other tissues, apoptosis is of paramount importance as a regulator of cell homeostasis and also serves as a potential mediator of tissue injury^ref. A number of lung diseases, such as idiopathic pulmonary fibrosis^{ref1, ref2}, acute respiratory distress syndrome (ARDS)^ref, chronic obstructive lung disease^ref, and bacterial pneumonia^ref, are characterized by extensive apoptosis within the alveolar epithelium. In addition, multiple pneumotoxins such as silica^{ref1, ref2}, bleomycin^ref, asbestos^ref, and paraquat^ref can induce apoptosis of various cells. Apoptosis plays a fundamental role in the distal airway remodeling during the transition from the canalicular to saccular stages during lung organogenesis^ref. The development of an effective alveolar-capillary interface requires extensive cell remodeling and is documented by extensive apoptosis of both epithelial and interstitial cells in both the gestational^ref and postnatal^ref periods. The Fas/Fas ligand (FasL) system has been implicated in apoptosis within the lung. It appears that many cells within the lung, including the alveolar epithelium, constitutively express Fas^{ref1, ref2}. Activation of Fas by intranasal administration of an agonistic antibody produced marked alveolar type II epithelial cell apoptosis and pulmonary inflammation in normal but not in Fas-deficient (lpr) mice^ref. In addition, soluble FasL was found in a bioactive form in the bronchoalveolar lavage fluid in patients during the development of acute respiratory distress syndrome (ARDS)^ref. At least one source of this FasL has been identified as neutrophils^ref, whose accumulation within the air spaces represents a hallmark of ARDS. Similarly, FasL was found in bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis but not controls^ref. Pulmonary fibrosis can also be mimicked in mice by administration of FasL^ref. Importantly, bleomycin-induced lung injury, a well-established model of fibrosis, was also attenuated in lpr and gld mice, which are deficient in Fas and FasL, respectively^ref. Lastly, the periods of lung organogensis characterized by high levels of apoptosis are also marked by increases in tissue expression of FasL^ref, an observation that strongly suggests that the Fas/FasL system serves to regulate developmental lung remodeling as well as tissue injury. Thus apoptosis within the lung contributes to both acute and chronic lung injury, as well as lung fetal and neonatal development.

Oxidative stress is one of the most common triggers of apoptosis. In addition, apoptosis is frequently accompanied by the generation of reactive oxygen species (ROS), resulting in part from cytochrome c (cyt c) departure from mitochondria and concomitant disruption of electron transport with enhanced generation of one-electron-reduced species of molecular oxygen within the cell^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10}. ROS represent attractive candidates for final common mediators of apoptosis, yet a specific role for ROS in the execution or resolution of the apoptotic program has not been established. Although effects of oxidative stress on the apoptotic machinery, including the caspases^{ref1, ref2}, and mitochondrial proteins forming the permeability transition pore (PTP)^{ref1, ref2,ref3} have been described, information on peroxidation of phospholipids and in particular on selective oxidation of their specific classes is scarce. This is mainly due to the fact that quantitative assays for oxidation of different classes of phospholipids are not readily available, which, in turn, is due in large part to a highly effective system of remodeling and repair of oxidatively modified phospholipids^ref that interferes with their accurate measurement. To characterize phospholipid oxidation during oxidative stress-induced apoptosis, a protocol based on metabolic labeling of cellular phospholipids with a natural oxidation-sensitive and highly fluorescent fatty acid, cis-parinaric acid (PnA), has been developed. This reagent has been extensively used in its free (nonesterified) form for structural measurements in membranes as well as in assays of oxidative stress in simple model systems^{ref1, ref2}. Metabolic labeling yields cells containing the major phospholipid classes [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol, diphosphatidylgycerol, and sphingomyelin (SPH)] fluorescently labeled with PnA and extremely low intracellular concentrations of free PnA^ref. Because free PnA is not available for phospholipid repair, resolution of major phospholipid classes by fluorescence HPLC can be used to quantify their oxidative damage (as a decreased content of fluorescent PnA residues in respective phospholipid classes). The level of PnA labeling of endogenous phospholipids (1–3mol%) is low enough to have minimal effects on cell viability and functions yet sufficient to permit quantitative detection of oxidative stress^ref. Importantly, the PnA-based assay can identify the selectivity of phospholipid oxidation on the basis of their polar head groups, and it is obviously independent of the fatty acid composition of phospholipids^{ref1, ref2}. Different phospholipids undergo nonrandom peroxidation during oxidant-induced apoptosis in a number of different cell lines. In particular, preferential oxidation of PS was typical of apoptosis induced by a number of oxidants, such as organic hydroperoxides, paraquat, and azo-initiators of peroxyl radicals. Most notably, there was a strong correlation between PS peroxidation and its externalization during apoptosis. In all cases when enhanced PS peroxidation occurred, PS externalization took place as well and vice versa; the lack or inhibition of preferential PS peroxidation during apoptosis was accompanied by the lack of PS externalization. The fundamental association of PS oxidation with apoptosis was strengthened by experiments in which a vitamin E homolog, 2,2,5,7,8-pentamethyl-6-hydroxy-chromane (PMC), was used. The lipophilic azo-initiator of radicals 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN) was employed to generate membrane-confined oxidative stress and induce apoptosis in HL-60 cells^ref. As an effective radical scavenger, PMC was able to completely protect all phospholipids against oxidation with the remarkable exception of PS. Furthermore, PMC failed to protect HL-60 cells against apoptosis following AMVN.

cell line	stimuli		apoptosis	PS externalization	PS oxidation
HL-60	oxidant-induced apoptosis	AMVNref	+	+	+
HL-60		AMVN + 2,2,5,7,8-pentamethyl-6-hydroxy-chromane (PMC)ref	+	+	+
HL-60		AMVN + NO·ref	+	+	-
HL-60		H2O2	+	+	+
HL-60		Cu-NTA + NO·ref	+	+	+
HL-60		Cu-NTAref	+	+	+
32D		paraquatref1, ref2	+	+	+
32D/Bcl2		paraquatref1, ref2	-	-	-
NHEK		cumene hydroperoxideref	+	+	+
dHL-60		PMAref	+	+	+
dHL-60		PMA + zVAD-fmkref	-	-	-
dHL-60		zymosanref	+	+	+
HL-60	nonoxidant-induced apoptosis	staurosporineref	+	+	+
NCl-H226		antisense-bcl2ref	+	+	+
Jurkat		anti-Fasref	+	+	+
A549		anti-Fas	+	+	+
PC12		neocarzinostatin (NCS)ref	+	+	+

The temporal sequence of PS oxidation and externalization on the cell surface is compatible with a causal link between these 2 events. Indeed, if this is the case, PS oxidation should occur within the plasma membrane where PS translocation events during apoptosis are known to occur. To address this issue, subcellular fractionation experiments were exerted in PnA-labeled cells challenged with tert-butyl hydroperoxide (t-BuOOH). t-BuOOH induced apoptosis and prominent PS oxidation in cells and different organelles. Most importantly, the highest rate of PS oxidation was detected in the plasma membrane compared with other organelles such as mitochondria, nuclei, lysosomes, and microsomes. The causative link between PS oxidation and apoptosis was further supported by experiments with DMSO-differentiated HL-60 cells showing inducible NADPH oxidase activity^ref. Activation of the NADPH oxidase by phorbol 12-myristate 13-acetate (PMA) or zymosan caused massive production of superoxide- and hydrogen peroxide (H₂O₂)-associated with oxidation of essentially all major phospholipids such as PC, PE, and PS. Exposure to PMA also induced apoptosis in these cells as evidenced by PS externalization on the cell surface, caspase activation, chromatin condensation, and nuclear and DNA fragmentation. All these effects were suppressed by inhibitors of the NADPH oxidase, diphenylene iodonium (DPI), or staurosporine. Remarkably, the pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone was able to significantly protect PS against PMA- (or zymosan-) induced oxidation, whereas oxidation of other phospholipids was insensitive to the inhibitor. The above data indicate that PS oxidation may be largely associated with the execution of apoptotic program. This association, however, is obscured during oxidant-induced apoptosis due to high background nonspecific oxidation. Several models of nonoxidant-induced apoptosis revealed that PS was again selectively oxidized compared with other more abundant phospholipids. In particular, decreased levels of Bcl-2 protein expression achieved via manipulating the level of bcl-2 mRNA translation in the squamous NSCLC line NCI-H226 by use of a synthetic antisense-bcl-2 oligonucleotide resulted in selective oxidation of PS in the subpopulation of cells with externalized PS. No significant differences in oxidation of cis-PnA-labeled PE and PC in cells were found after treatment with nonsense or antisense-bcl-2 oligonucleotides^ref. Similarly, by exposing HL-60 cells to staurosporine, a protein kinase inhibitor without direct prooxidant activity, apoptosis was induced in HL-60 cells without triggering confounding nonspecific oxidation reactions. PS underwent preferential oxidation at an early stage of apoptosis, whereas the most abundant phospholipid, PC, and GSH, the most abundant cytosolic thiol, remained unoxidized^ref. Finally, Fas triggering of Jurkat T lymphocytes resulted in oxidative stress with specific PS oxidation and externalization, whereas Raji cells, which are defective for PS exposure, did not undergo PS oxidation^ref. Expectedly, anti-Fas triggering of PS oxidation/externalization was accompained by phagocytosis of apoptotic Jurkat cells (but not Raji cells) by J774A.1 macrophages. Because several recent reports suggest that Fas/FasL are implicated in apoptosis within the lung, the human pulmonary adenocarcinoma A549 cell line was utilized as a convenient model for alveolar epithelial cells to evaluate the effect of Fas-triggered apoptosis on membrane phospholipid oxidation : induction of apoptosis in IFN-g-pretreated A549 cells by anti-Fas mAb caused specific PS oxidation along with its externalization. In sum, 3 important features of PS oxidation during apoptosis have been established : first, the preferential oxidation of PS is observed only in intact living cells undergoing apoptosis and not in cell-free liposome preparations incubated with oxidants. Second, PS oxidation occurs early during execution of the apoptotic program and precedes the appearance of such hallmarks of apoptosis as DNA fragmentation and, most importantly, PS externalization. Finally, PS peroxidation is blocked in cells overexpressing antiapoptotic gene products such as Bcl-2 and is sensitive to pancaspase inhibitors^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}. Selective PS oxidation suggests that there may be specific catalytic mechanism(s) responsible for the predominant oxidation of this particular class of phospholipids. One possible mechanism relies on the involvement of cyt c released from mitochondria into the cytosol during apoptosis. Cyt c release, an early and common marker of apoptosis, is involved in the formation of the so-called apoptosome complex and the subsequent activation of caspases downstream of mitochondria^ref. This proapoptotic role of cyt c seems to be redox independent and does not require the presence of the heme moiety^{ref1, ref2}. However, a significant part of cyt c is not released as an apoprotein but rather contains heme with its redox-active iron^{ref1, ref2}. Moreover, as cyt c is a basic protein (pI 10.3) that is positively charged at neutral pH^ref, it may be predisposed to interact electrostatically with negatively charged phospholipids such as PS^{ref1, ref2}. In fact, it has been demonstrated that cyt c effectively binds to negatively charged PS-containing membranes^ref. Electrostatic interaction of cyt c with negatively charged sites of membranes induces disruption of the Met⁸⁰-Fe(heme) coordination bond and partial unfolding of the protein globule, thus facilitating orientation of the heme moiety along the membrane surface^ref. Disturbance of Met⁸⁰-Fe(heme) coordination renders Fe more catalytically redox active, while unfolding of the protein positions its heme catalytic site closer to phospholipid targets. Oxidized cyt c is less stable, and the energy of its unfolding is lower than for the reduced form^ref, suggesting that PS in the inner leaflet of the plasma membrane may preferentially interact with the oxidized form of cyt c. Importantly, departure of cyt c from mitochondria is accompanied by a massive production of H₂O₂, which may promote oxidation of cyt c heme^ref. Thus one may speculate that, once released from mitochondria, cyt c becomes oxidized, binds to PS-rich lipid rafts on the inner surface of plasma membrane, and unfolds to expose its redox-active heme-iron moiety to phospholipids, particularly to PS, the electrostatically most attractive phospholipid species. As a result, PS may be more susceptible to cyt c-catalyzed oxidation than other phospholipids during apoptosis. In cells committed to programmed death after exposure to proapoptotic signals, mitochondrial permeability transition and activation of pores take place. 2 functionally important proteins, the caspase coactivator cyt c and the caspase-independent death effector apoptosis-inducing factor (AIF), are released from mitochondria into the cytosol. Disruption of mitochondrial electron transport is accompanied by production of superoxide; the latter dismutates to H₂O₂, which can readily diffuse into the cytosol. Electrostatic interactions of positively charged cyt c with negatively charged PS may facilitate cyt c-catalyzed generation of highly reactive oxidants (e.g., oxo-ferryl species) in close vicinity of PS, thus providing for its selective peroxidation. PSox can contribute to poisoning of aminophospholipid translocase (APT), thus providing for its own externalization along with PS. Alternatively, PSox may undergo effective transmembrane diffusion. Finally, PSox and PS synergistically interact as an "eat-me" signal for phagocytic recognition of apoptotic cells. This effect of PSox may be realized through its interaction with one of the known macrophage receptors, such as PS receptor (PSR), CD36, LOX-1, or with an as-yet unidentified PSox receptor (PSoxR). Finally, antioxidants by blocking PS oxidizing pathway(s) can affect PS externalization and subsequent clearance of apoptotic cells.

Cyt c as a catalyst for selective PS oxidation during apoptosis: release of cyt c from mitochondria into the cytosol is one of the early and common events in apoptosis. In the cytosol, cyt c participates in the activation of the caspase cascade. As a basic protein (pI 10.3), it is positively charged at neutral pH and can electrostatically interact with negatively charged phospholipids such as PS. As a result, redox (prooxidant) catalytic activity of heme-containing cyt c may be directed toward selective PS oxidation. Production of superoxide and H₂O₂ by disrupted mitochondrial electron transport facilitates formation of reactive oxidants such as oxo-ferryl species of heme-containing cyt c in the immediate vicinity of PS thus providing for PS oxidation. PSox can be externalized and enhance externalization of PS

There are several experimental findings that are compatible with the model outlined above. In one series of experiments, incorporation of cyt c into PnA-labeled HL-60 cells (in the presence of exogenously added t- BuOOH) resulted in preferential oxidation of PS compared with other phospholipids^ref. Furthermore, in cell-free model systems, PS proved to be selectively oxidized by a cyt c/ascorbate/H₂O₂-catalytic system compared with PC^ref. In another series of experiments, researchers used the DP-1 peptide, which is composed of 2 functional domains: a protein transduction domain, PTD-5 (a 12-mer peptide sequence RRQRRTSKLMKR from M13 phage library), and an antibiotic peptide, KLA [(KLAKLAK)₂]. PTD-5 is used to guide KLA to target cells and allow for its internalization. PTD-5 is positively charged due to a high content of arginine and lysine residues (70%) and causes efficient and rapid internalization of conjugated proteins into cells in vitro and in vivo^ref. KLA, an antimicrobial peptide, is designed to target and disrupt bacterial cells, as well as mitochondria in eukaryotic cells^{ref1, ref2}. Therefore, the PTD-5/KLA conjugate (DP-1) is able to preferentially disrupt mitochondria and induce release of cyt c but spare damage to the plasma membrane and other organelles in cells. Incubation of Jurkat cells with DP-1 peptide caused a rapid (within 5 min) release of cyt c from mitochondria into cytosol. Importantly, cyt c released from mitochondria could be completely recovered in the cytosolic fraction. Furthermore, DP-1-induced cyt c release was accompanied by a > 4-fold increase in H₂O₂ production by Jurkat cells. Next, using the PnA-based assay, PS was the only phospholipid that was significantly oxidized after incubation of Jurkat cells with 10 µM DP-1 DP-1-induced PS oxidation was accompanied by significant externalization of PS in Jurkat cells and enhanced recognition and phagocytosis of these cells by J774A.1 macrophages. Together, these data suggest that release of cyt c may be involved in PS oxidation. The plasma membrane, the barrier between intra- and extracellular milieus, plays a pivotal role in the communication of cells with their environment. The majority of aminophospholipids (PE and PS) are predominantly confined to the inner leaflet of the plasma membrane, whereas choline-containing phospholipids (PC and SPH) are localized mainly in its outer leaflet^{ref1, ref2, ref3, ref4, ref5}. This phospholipid asymmetry is maintained by an ATP-dependent aminophospholipid translocase that specifically transports PS and PE from the outer to the inner leaflet^ref. Aminophospholipid translocation is Ca²⁺ inhibitable and sensitive to the sulfhydryl-reactive agent N-ethylmaleimide (NEM), as well as to vanadate, an inhibitor of P-type ATPases. The molecular identity of this "flippase" is not firmly established, although a candidate P-type ATPase termed ATPase II (ATP8A1 / aminophospholipid transporter (APLT), ATP8B3, ATP10A / ATPase type IV, phospholipid transporting (P-type)) that possesses phospholipid transporting properties has been identified^{ref1, ref2, ref3, ref4, ref5, ref5, ref6}. Loss of membrane phospholipid asymmetry with subsequent externalization of PS is known to be an important signaling mechanism, e.g., to stimulate the coagulation cascade during platelet activation and to mediate cell recognition by macrophages^ref. This process is believed to be mediated by a Ca²⁺-activated phospholipid scramblase that facilitates the bidirectional movement of all classes of phospholipids across the the lipid bilayer^ref. Zhou and coworkers^{ref1, ref2} identified a 37-kDa protein in platelets with scramblase activity and have demonstrated a correlation between the expression of this scramblase and the Ca²⁺ ionophore-induced externalization of PS. However, this protein is normal in Scott syndrome patients whose blood cells are defective for scramblase activity and PS exposure^{ref1, ref2}, suggesting that additional molecules are required for scramblase activation. Moreover, red blood cells from scramblase null mice normally mobilize PS to the surface upon Ca²⁺ ionophore stimulation^ref. PS externalization is considered a general phenomenon in cells undergoing apoptosis^{ref1, ref2}, although a few instances of apoptosis in the absence of PS exposure have also been described^{ref1, ref2}. PS exposure is dependent on extracellular Ca^{2+ref1, ref2, ref3} and occurs downstream of the activation of caspases^{ref1, ref2, ref3}. In addition, previous studies have indicated that PS exposure during apoptosis is dependent on the concomitant inhibition of the aminophospholipid translocase and activation of the phospholipid scramblase^{ref1, ref2}. However, recent data suggest that scramblase expression is not a critical determinant of apoptotic PS exposure^{ref1, ref2}. Moreover, the inhibitor of the aminophospholipid translocase, NEM, can induce PS exposure in the absence of other indices of apoptosis^{ref1, ref2}, and the NEM-induced redistribution of PS in Raji cells is sufficient to trigger macrophage removal of these cells^ref. Together, these findings serve to underscore the role of aminophospholipid translocase inhibition for the outward movement of PS during apoptosis. Importantly, although disturbances of phospholipid asymmetry during apoptosis are caspase dependent, neither aminophospholipid translocase nor scramblase has been reported as a direct target of caspases, the major proteolytic executors of apoptosis^{ref1, ref2}. Therefore, alternative mechanisms affecting these enzymatic activities must be responsible for disturbances of phospholipid asymmetry during apoptosis. Intracellular ATP can modulate aminophospholipid translocation and PS exposure during Fas-mediated apoptosis, irrespective of the scramblase status of the cell^ref, and we have observed that Bcl-2 overexpression maintains intracellular ATP levels and abrogates PS exposure in Fas-triggered SKW6.4 cells. In addition, the aminophospolipid translocase is sensitive to oxidative and nitrosative modification of its SH groups^{ref1, ref2}, and it is therefore tempting to speculate that oxidative stress may also play a role in translocase inhibition and the subsequent loss of phospholipid asymmetry. Importance of lipid rafting for the externalization of PS and its recognition. Apoptosis-associated exposure of PS on the cell surface is only one feature typical of the global and complex biochemical (flipping of SPH, redistribution of cholesterol) and biophysical (changes of fluidity, segregation of lipids, and formation of microdomains) rearrangements of the plasma membrane during apoptosis culminating in its dramatic blebbing and vesiculation^{ref1, ref2}. Lipid rafts are specialized membrane subdomains that have a high cholesterol and sphingolipid (50 and 20%, respectively) content and are organized in a tightly packed, liquid-ordered manner^{ref1, ref2}. Various proteins (e.g., the multifunctional scavenger receptor CD36) selectively partition into detergent-resistant lipid rafts, suggesting that clustering of proteins within rafts is a process regulated by specific lipid-protein interactions^ref. Aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling. Recent studies have provided evidence that raft integrity is essential for the plasma membrane redistribution of PS in Ca²⁺ ionophore-stimulated tumor cells^ref. It has been speculated that the formation of rafts also participates in PS exposure during apoptosis^ref. Such aggregation of PS molecules may facilitate their subsequent recognition by one or more PS receptor(s) (PSR) on the macrophage surface. Interestingly, clustering of ced-1, a putative PSR in Caenorhabditis elegans, is seen in response to cell corpse recognition in the nematode system^ref. Although it is not known whether PS oxidation is involved in PS aggregation and formation of rafts in the plasma membrane during apoptosis, it is noteworthy that aggregation of phospholipid hydroperoxides resulting in the formation of their clusters has been documented. Annexin V colocalizes with markers of lipid rafts in the outer membrane of activated, nonapoptotic B cells, suggesting that PS exposure can occur in specific membrane microdomains in the absence of cell death^ref. Moreover, mature B cells expose PS on their surface, where it colocalizes with antigen receptors and forms caps that are required for receptor-mediated signaling events that trigger Ig production^ref. Similarily, transient, nonapoptotic exposure of PS is seen during development of skeletal and heart muscle and has been suggested to be essential for myotube formation in mice^ref. Finally, normal macrophages themselves were reported to display PS on their surface, and this was suggested to be required for phagocytosis of PS-expressing target cells^ref. These observations beg the question of how cells that express the common PS-dependent eat-me signal can escape macrophage recognition. One may speculate that the functional outcome of PS externalization may ultimately depend on the density of PS on the cell surface. A low or intermediate level of PS or transient exposure of PS may not suffice to trigger clearance, whereas more extensive externalization of PS during apoptosis may reach a threshold necessary for phagcoytosis to occur (see below). Alternatively, viable nonapoptotic PS-positive cells may fail to express additional necessary cofactors, such as chemotactic mediators or accessory surface ligands (such as oxidized PS), required for the stimulation of macrophages. Evaluations of amounts of externalized PS in nonapoptotic and apoptotic cells. Because apoptosis is accompanied by PS oxidation in the plasma membrane, two different populations of PS, nonoxidized PS (PS) and oxidized PS (PSox), are likely to be present in the outer membrane leaflet of apoptotic cells. These two PS species may behave differently with regard to their topography in the membrane as well as their effects on enzymatic and nonenzymatic pathways involved in the maintenance of PS asymmetry across the plasma membrane. PSox may act as a poison or "suicide substrate" for the aminophospholipid translocase, thus inhibiting translocation of both PSox and PS. This would result in the appearance of both PS and PSox on the surface of the plasma membrane. Alternatively, PSox could be preferentially externalized during apoptosis as a result of poor substrate recognition by the translocase and/or a high rate of spontaneous (nonenzymatic) transbilayer flip-flop. Methods for the quantitative assessment of PS content on the surface of apoptotic cells are clearly needed to determine the levels of native PS, PSox, and potentially other molecular species of PS and the mechanism of their redistribution within the plasma membrane during apoptosis. Although annexin V-based assays for PS externalization have been extensively used in numerous studies, the results are usually interpreted in terms of distribution of cell populations with an arbitrary (above threshold) amount of externalized PS rather than the absolute amount (concentrations) of PS expressed on the cell surface. Unfortunately, quantification of PS on apoptotic cells has not received significant attention. Indeed, since the pioneering work by Fadok and colleagues^ref, who discovered PS externalization in apoptotic murine thymocytes using chemical derivatization with fluorescamine, a nonpermeable reagent for primary amines, there are only few examples of PS quantitative measurements (e.g., in the human leukemia T cell line Jurkat and the human leukemia HL-60 cell line). However, neither the fluorescamine-based assay nor the annexin V-based flow cytometric assay permits the quantitative estimation of amounts of PS on the surface of nonapoptotic cells. Using a modification of the annexin V method, the annexin V iron-beads/electron paramagnetic resonance assay, we were recently able to quantify PS on the surface of Jurkat cells and HL-60 cells. Apoptotic HL-60 cells and Jurkat cells externalized up to 25–280-fold more PS than nonapoptotic controls. This suggests that PS is a prominent signal for clearance of apoptotic cells by macrophages
PS expression on the surface of naïve and apoptotic cells :

			PS externalization, pmol/106 cells		incubation conditions
			HL-60 Cells	Jurkat Cells
annexin V-iron bead EPR assay	control		1.1±0.3	0.8±0.6
	apoptotic	anti-FAS antibody	N/A	239±18	0.5 µg/ml, 4 h
		staurosporine	27±13	N/A	1 µM, 4 h
		camptothecin	22±18	237±62	10 µM, 4 h for HL-60 cell, 50 µM, 4 h for Jurkat cell
fluorescamine-based assay	control		6.1±2.4	2.2±1.9
	apoptotic	AMVN	129.1±21.5		500 µM, 2 h
		anti-FAS antibody		243±27	0.25 µg/ml, 2 h

The importance of PS on the cell surface for recognition and phagocytosis by macrophages was first demonstrated by Schroit and colleagues^{ref1, ref2} in the early 1980s in experiments using red blood cells with artificially manipulated levels of exogenous PS. These investigators hypothesized that recognition of PS-exposing cells by macrophages involves specific ligand-receptor interactions^ref. Further studies demonstrated that activated monocytes are able to bind different tumor cell lines with elevated levels of PS, but not normal human keratinocytes cells with relatively low PS content^ref. These results imply that a threshold of PS expression may exist for macrophage recognition of PS-externalizing cells^ref. To further explore this hypothesis, we enriched the external leaflet of the plasma membrane of nonapoptotic Jurkat cells and HL-60 cells with known amounts of exogenous PS and determined the sensitivity of macrophages for PS on the surface of the target cells. The dependence of the phagocytic capacity of macrophages on the amounts of PS integrated into the outer leaflet of plasma membrane in Jurkat cells or HL-60 cells unequivocally demonstrated a nonlinear response with a sensitivity threshold required to initiate macrophage recognition of externalized PS. Hence, at least a 5–10-fold increase of the PS content in the outer leaflet of plasma membrane was required to trigger macrophage recognition and uptake. During apoptosis, e.g., induced by the chemotherapeutic agent camptothecin, both cell lines expressed externalized PS in amounts far in excess of the recognition threshold and were thus effectively phagocytosed. Many receptors have been implicated in the removal of dying, apoptotic cells by macrophages. Not only do different populations of phagocytes use discrete receptors, but a single phagocyte may express a number of receptors that cooperate in the ingestion of their prey^ref. These different receptors include the class A scavenger receptor^{ref1, ref2}, V3-integrin (vitronectin receptor)^{ref1, ref2}, CD68^ref, CD14^ref, lectin-like oxidized low-density lipoprotein receptor, and CD36^{ref1, ref2}. An interesting alternative point of view is that CD31 acts as a cell surface molecule that normally prevents phagocyte ingestion of viable cells by transmitting "detachment" signals^ref. CD31-mediated detachment is disabled in apoptotic cells by an unknown mechanism, resulting in the promotion of tethering of cell corpses to phagocytes. In addition, serum proteins such as b₂-glycoprotein I^{ref1, ref2} and C1q, the first component of complement ^ref, bind to apoptotic cells and enhance their uptake. Many of the above receptors can bind PS, but not all of them are specific for this phospholipid^{ref1, ref2}. Furthermore, activated macrophages were recently reported to secrete a glycoprotein, milk fat globule epidermal growth factor 8, that specifically binds to PS on apoptotic cells and to V3-integrin on the macrophage surface, thus serving as a "molecular bridge" between the 2 cells^ref. Regardless of the receptors engaged or disengaged in phagocytosis, ingestion does not occur in the absence of PS exposure^{ref1, ref2}. Specifically, recognition of surface PS was reported to be dependent on the presence of the so-called PSR, a recently cloned receptor that is expressed by phagocytes and mediates pinocytosis and initiates uptake of apoptotic cells^{ref1, ref2, ref3}. Indeed, the requirement for PS expression and the ensuing ligation of PSR on phagocytes by PS on the apoptotic cell surface may be essential to signal uptake of cells that are tethered to phagocytes via other receptors^ref. However, additional signals, such as oxidative changes, may also be required for the engulfment of target cells^ref. In fact, many of the receptors implicated in phagocytosis of apoptotic cells can strongly bind oxidized phospholipids^{ref1, ref2}, which arise during apoptosis and provide additional ligands for recognition receptors^{ref1, ref2}. Oxidized epitopes on the surface of apoptotic cells may thus act as important signals for the recognition of target cells by macrophages^{ref1, ref2}. In particular, C-reactive protein, a component of the innate immune response, was shown to bind to oxidized PC species on the surface of "late" apoptotic (propidium iodide-positive) cells^ref. Moreover, Podrez et al.^{ref1, ref2} recently described a novel family of oxidized PC homologs that were able to act as a ligands for the scavenger receptor CD36 and promote macrophage foam cell formation. Because plasma membrane PS may be a specific target for oxidation, it is tempting to speculate that a combination of PS and PSox may be essential for recognition and uptake of apoptotic cells. In support of this notion, inhibition of PS oxidation in cells during apoptosis has been demonstrated to block phagocytosis of Jurkat cells and HL-60 cells by macrophages^{ref1, ref2}. Moreover, integration of PSox along with PS into plasma membrane of nonapoptotic cells significantly stimulated their phagocytosis compared with the incorporation of similar amounts of native PS alone^ref. In addition, liposomes containing PSox acted as potent inhibitors of phagocytosis of apoptotic cells (anti-Fas-triggered Jurkat cells and t-BuOOH-treated HL-60 cells)^ref. Together, these findings indicate that PSox, indeed, may act as an important signal on the cell surface that can act alone or in combination with native PS to facilitate recognition of apoptotic cells. Nevertheless, many questions remain regarding the role of PSox in the signaling for engulfment. For example, what is the fraction of PSox vs. PS on the cell surface during apoptosis? Can other oxidized phospholipids such as PCox, PEox, etc. synergistically interact with PS to facilitate recognition of apoptotic cells? What are the actual concentrations of PCox, PEox, and other oxidized phosopholipids compared with PSox on the cell surface during apoptosis? Interestingly, our preliminary experiments indicate that PSox is capable of markedly reducing the threshold for recognition of PS-containing cells by macrophages. Another important question relates to the type of receptors involved in recognition of PS and PSox. It seems likely that recognition of PS and PSox involves different receptor(s). Indeed, anti-PSR antibody, but not anti-CD-36 antibody, was able to inhibit phagocytosis of Jurkat cells with PS integrated into their plasma membrane. In contrast, both anti-PSR antibody and anti-CD36 antibody were effective in suppressing phagocytosis of Jurkat cells enriched with both PS and PSox; anti-CD36 antibody was also efficient in suppressing the engulfment of Fas-triggered Jurkat cells, which display both PSox and its nonoxidized counterpart on the cell surface. These data imply that CD36 and PSR might cooperate to recognize oxidized PS. In conclusion, selective oxidation and externalization of PS in the plasma membrane of apoptotic cells likely creates conditions whereby oxidized PS on the external surface of the cell could act as a preferred ligand (or eat-me signal) for certain macrophage receptors, including scavenger receptors such as CD36, resulting in the recognition and disposal of cell corpses. The potentially important signaling function of PS oxidation in the plasma membrane and the subsequent externalization of PSox on the cell surface suggest that antioxidants theoretically could play an unusual role in the regulation of apoptosis and phagocytosis of apoptotic cells. Thus if PS oxidation is essential for its externalization and, furthermore, if PSox acts as an additional stimulatory signal facilitating recognition and engulfment of apoptotic cells, then inhibition of PS oxidation by antioxidant enzymes and/or water- and lipid-soluble antioxidants might interfere with the execution of these critical functions and, hence, with the resolution of the apoptotic process. Obviously, total nonspecific prevention of oxidative stress during oxidant-induced apoptosis by antioxidants via blocking the initiation of apoptotic program is a trivial and highly predictable effect that has been documented in a number of reports^{ref1, ref2}. Therefore, models of nonoxidant apoptosis, where oxidative stress functions only as a specific component of the execution of apoptotic program, are preferable for studies of antioxidant effects. In particular, observations related to PS-specific effects of antioxidants may be of considerable interest. Antioxidant enzymes can indeed modulate PS oxidation-dependent signaling pathways in apoptosis. This was demonstrated in experiments with Fas-triggered apoptosis in Jurkat cells^ref. Anti-Fas antibody triggered selective oxidation of PS accompanied by PS externalization, caspase activation, and recognition and phagocytosis of apoptotic cells by several classes of macrophages. Remarkably, high doses of the antioxidant enzymes superoxide dismutase (SOD)/catalase (50 U/ml of each) were able to inhibit oxidative stress (e.g., production of superoxide and H₂O₂) and block PS oxidation and recognition/phagocytosis of apoptotic target cell by J774A.1 macrophages without affecting other biomarkers of apoptosis, such as PS externalization, caspase-3 activation, and nuclear condensation. An SOD mimetic, Mn(III) tetrakis (5,10,15,20-benzoic acid) porphyrin, protected T cells after activation through Fas/TNF- (i.e., death receptor)-independent pathways^ref. This protection, however, was due to decreased mitochondrial damage and subsequent caspase-dependent DNA fragmentation. It seems, therefore, that ROS may be differentially involved in signaling and execution pathways during apoptosis depending on the initial triggering mechanisms. Not only antioxidant enzymes but also low-molecular-weight chain-breaking antioxidants can affect apoptotic pathways by inhibiting PS oxidation. For example, an effective lipid antioxidant, etoposide (VP-16), at pharmacologically relevant concentrations (50 µM) was able to completely block PS oxidation in HL-60 cells during H₂O₂-induced apoptosis. Under these conditions, etoposide inhibited PS externalization in HL-60 cells as well as their phagocytosis by J774A.1 macrophages. It is important that effects of antioxidants on PS oxidation and subsequent PS-dependent pathways are studied and determined specifically during apoptosis rather than when both apoptotic and necrotic mechanisms are realized concomitantly. In this regard, Shacter and colleagues^{ref1, ref2}reported that high concentrations of H₂O₂ inhibited phagocytosis of apoptotic cells upon etoposide treatment, largely due to the shifting of cell death from apoptosis to necrosis. Vitamin E (a-tocopherol) is one of the major natural lipid-soluble chain-breaking antioxidants of membranes. The effects of vitamin E on Fas-triggered oxidation of PS, apoptosis in Jurkat cells, and their phagocytosis by J774A.1 macrophages were experimentally determined. Substantial inhibition of Fas-induced phospholipid oxidation could be achieved only at relatively high pharmacological concentrations of vitamin E. Complete inhibition of Fas-induced PS oxidation was observed only when vitamin E levels in cells were >20-fold in excess of those in nonsupplemented cells. At these high doses, however, vitamin E did not affect the outcome of apoptosis as shown by PS externalization and nuclear morphological alterations. No changes in the effectiveness of phagocytosis of apoptotic cells occurred in the presence of vitamin E. Notably, physiologically relevant levels of vitamin E were not able to completely block PS oxidation. It should be mentioned, however, that the effects of vitamin E on phagocytosis may be realized through its antioxidant-independent mechanisms of macrophage activation, such as induction of the expression of cell-cell adhesion molecules^{ref1, ref2}. Thus physiological levels of antioxidants are not likely to be sufficient to completely block PS oxidation during apoptosis and hence to interfere with PS-dependent pathways of phagocytosis. High pharmacological doses of antioxidants, however, are able to cause inhibition of PS oxidation sufficient to affect PS externalization (etoposide) or phagocytosis (SOD/catalase). Antioxidants are commonly believed to be effective anti-inflammatory agents^ref. However, it is important that their use at high doses be considered carefully, as their specific mechanism of action predicts a potential effect as inhibitors of PS-dependent pathways in apoptosis and phagocytosis of apoptotic cells, thus precluding the noninflammatory removal of dying cells. Neutrophils are an important line of host defense against invading microorganisms, and the production of ROS in these cells is an essential step in the killing of ingested bacteria. The apoptotic death of neutrophils and their subsequent engulfment by macrophages is believed to be a critical component in the resolution of inflammation, as this serves to remove these cells from the inflammatory site with minimal damage to surrounding tissues^{ref1, ref2}. Conversely, a mismatch between the rate of apoptosis and the rate of phagocytic clearance of apoptotic cells may underlie detrimental inflammatory responses, as shown in, e.g., mice receiving agonistic anti-Fas antibodies^ref. These animals die from hepatic failure as a result of massive apoptosis of hepatocytes and fulminant inflammation in the liver, due most likely to "secondary" necrosis of unengulfed cells. The harnessing of apoptotic mechanisms involved in the resolution of inflammation and restitution of tissue homeostasis may thus yield novel therapeutic strategies in conditions of excessive inflammation. Cystic fibrosis (CF) is an inflammatory disease of the lung characterized by a sustained influx of inflammatory cells into the airways and release of proteases from these cells^ref. The fact that inflammation is persistent and that necrotic (or postapoptotic) cells accumulate in the airways of CF patients suggests that the normal mechanism for removal of effete cells is impaired. Indeed,  an abundance of unengulfed apoptotic cells was demonstrated in the airways of CF as well as non-CF bronchiectasis patients^ref. These investigators also provided evidence that neutrophil elastase, an intracellular protease that is released by inflammatory cells into the airways during inflammation, cleaves the PSR on the surface of phagocytes, thus contributing to the disruption of apoptotic cell clearance. Consequently, therapies that augment macrophage engulfment of apoptotic cells, for instance by targeting the PS-dependent pathway of cell clearance, may be envisaged for conditions of pulmonary decline due to excessive inflammation. Another example of chronic inflammation is the rare hereditary disease known as chronic granulomatous disease (CGD), which is characterized by severe and sometimes fatal infections, including fungal or bacterial pneumonia^ref. The basic defect arises from an inability of neutrophils to generate superoxide and H₂O₂ due to mutations in the membrane-bound NADPH oxidase. Neutrophils possess both caspase-dependent and oxidant-dependent modes of PS exposure, which are employed during constitutive apoptosis and activation-induced (nonapoptotic) death, respectively^{ref1, ref2}. Whether PS is also oxidized in neutrophils undergoing apoptotic vs. nonapoptotic cell death is undetermined yet. Nevertheless, our studies of CGD neutrophils demonstrated that although these cells exhibit a normal apoptotic response, with caspase activation and plasma membrane exposure of PS, they fail to externalize PS when incubated with the potent neutrophil activator PMA^ref. In addition, our recent studies using DMSO-differentiated, neutrophil-like HL-60 cells as well as neutrophils from healthy donors confirm that both PMA and opsonized zymosan beads, a more physiological stimulator of the NADPH oxidase, are capable of triggering ROS-dependent, DPI-inhibitable PS externalization^ref. Concomitant oxidation of PS was also observed in the HL-60 model. These data thus provide evidence for the involvement of NADPH-derived ROS in the oxidation and externalization of PS. H₂O₂-dependent PS externalization and macrophage uptake of neutrophils ingesting Staphylococcus aureus were documented^ref. It appears reasonable to speculate that the failure to activate this mode of PS exposure in vivo could result in defective clearance of cells by macrophages and hence contribute to the formation of the characteristic granulomatous lesions and subsequent tissue destruction evidenced in CGD patients. The augmentation of PS- and/or PSox-dependent cell clearance may serve as a therapeutic approach in CGD and related granulomatous disorders. Molecules other than PS and PSox may also contribute to the removal of effete cells during inflammation. For instance, ligation of the cell surface adhesion molecule CD44 is known to promote the uptake of apoptotic neutrophils but not of apoptotic lymphocytes^ref. Mice deficient for CD44 exhibit a >10-fold increase in unengulfed apoptotic cells in the lung tissue after bleomycin treatment compared with wild-type animals^ref. Importantly, lack of CD44 resulted in increased mortality from lung injury due to unremitting inflammation, thus serving to underscore the importance of clearance of apoptotic neutrophils in the resolution of lung inflammation. In view of the above observations concerning the importance of PS oxidation in the plasma membrane during apoptosis, it seems prudent to reconsider the clinical utilization of antioxidants, believed to act as promising therapeutics in the regulation of inflammatory responses. Thus it will be important to establish regimens and conditions for antioxidant treatments that do not affect important PS oxidation signaling events in apoptotic cells and hence interfere with macrophage recognition of such cells. Conversely, one can envision that directed and targeted delivery of PSox to the surface of damaged cells can be used to enhance their safe clearance and may be useful for limiting the inflammatory response in the lung and in other tissues. In summary, generation of ROS is an integral component of the apoptotic program and results in the preferential oxidation of PS in the plasma membrane of the dying cell. Oxidized PS, in turn, serves as an important signal through which macrophages recognize and eliminate apoptotic cells. Oxidative stress within the apoptotic cell may promote this clearance process either via enhanced externalization of oxidized PS on the surface of apoptotic cells and/or through more effective recognition of apoptotic cells exhibiting PSox (along with its nonoxidized counterpart) on their surface. Furthermore, we speculate that the mitochondrial release of cyt c during apoptosis is critically involved in the selective oxidation of PS and its subsequent externalization. Regardless of the specific mechanism(s) involved, the final outcome of macrophage recognition of PS and Psox is the nonphlogistic disposal of potentially harmful cells, e.g., at sites of inflammation. In this context, the possibility that antioxidants capable of inhibiting PS oxidation might interfere with PS externalization and/or its recognition by macrophages needs to be carefully considered. Nevertheless, as outlined in the present review, apoptosis-dependent, mitochondrially derived oxidative stress is mechanistically linked with the oxidation of PS and the stimulation of PS-dependent signaling pathways culminating in the disposal of cells by macrophages^ref.
Oxidation of low density lipoprotein (LDL) generates a variety of oxidatively modified lipids and lipid-protein adducts that are immunogenic and proinflammatory, which in turn contribute to atherogenesis. Cells undergoing apoptosis also display oxidized moieties on their surface membranes, as determined by binding of oxidation-specific monoclonal antibodies. In the present paper, we demonstrated by mass spectrometry that in comparison with viable cells, membranes of cells undergoing apoptosis contain increased levels of biologically active oxidized phospholipids (OxPLs). Indeed, immunization of mice with syngeneic apoptotic cells induced high autoantibody titers to various oxidation-specific epitopes of oxidized LDL, including OxPLs containing phosphorylcholine, whereas immunization with viable thymocytes, primary necrotic thymocytes, or phosphate-buffered saline did not. Reciprocally, these antisera specifically bound to apoptotic cells through the recognition of oxidation-specific epitopes. Moreover, splenocyte cultures from mice immunized with apoptotic cells spontaneously released significant levels of T_h1 and T_h2 cytokines, whereas splenocytes from controls yielded only low levels. Tthe OxPLs of apoptotic cells activated endothelial cells to induce monocyte adhesion, a proinflammatory response that was abrogated by an antibody specific to oxidized phosphatidylcholine. Apoptotic cell death generates oxidatively modified moieties, which can induce autoimmune responses and a local inflammatory response by recruiting monocytes via monocyte–endothelial cell interaction^ref.

The swift recognition and the phagocytosis of apoptotic cells are the object of intense investigation^{ref1, ref2}. However, the fate of the antigens they contain and the constrains limiting their immunogenicity in vivo are scarcely understood. Apoptotic cell antigens enter the MHC class I pathway of the phagocyte via the cytosol and become available for recognition by MHC-restricted CTLs. After engulfment of apoptotic cells, monocytes/macrophages release antiinflammatory cytokines, such as IL-10 and TGF-ß^ref. DCs that phagocytosed virus-infected apoptotic cells productively activate (cross-prime^ref) virus-specific CTçs^ref. DCs are unusually efficient in processing internalized dying cells for presentation to MHC class I– and class II–restricted T lymphocytes. Accordingly, DCs that internalized dying tumor cells efficiently cross-activate tumor-specific T cells, both in vitro and in vivo^ref. The presence of monocytes/macrophages, which release factors that prevent the maturation and the function of DCs, abrogates the cross-priming of T cells by phagocytosing DCs^ref. The in vivo immunogenicity of the highly tumorigenic Rauscher virus–induced H-2^b RMA lymphoma dramatically abates after apoptosis induction^ref. The efficacy of RMA lymphoma cells as vaccines was partially rescued in IL-10 knockout mice^ref. Moreover, annexin V (AxV) hindering the recognition of exposed phosphatidylserine (PS) restored the ability of dying xenogeneic cells to evoke antibody responses in vivo^ref. Altogether, the results suggest that dying cells administered in vivo cause the secretion of factors that in turn restrict their immunogenicity (immunosuppressive clearance). It is well established that PS serves as a recognition signal for the clearance of apoptotic cells. The redistribution of PS both on phagocyte and prey is involved^ref. A well-characterized receptor mediates the PS-dependent uptake of apoptotic cells (PS receptor)^ref, engaging a signaling pathway crucial for the rearrangement of phagocytes' actin-based cytoskeleton with eventual engulfment of the corpse. The activation of this receptor induces the release of immunosuppressive cytokines^ref. Accordingly, animals lacking PS receptor accumulate uncleared apoptotic cells at the periphery and targeted mice develop tissue inflammation^{ref1, ref2, ref3}. AxV, which binds to exposed PS residues in vitro and in vivo, inhibits apoptotic cell uptake by monocytes/macrophages most likely through interference with the availability of PS for recognition^{ref1, ref2, ref3, ref4}. Scavenger cells recognize exposed moieties on dying cells. The outcome is the swift phagocytosis of the corpses, which prevents secondary necrosis and the ensuing tissue damage. The recognition of exposed PS via its receptor^ref triggers the release of immunosuppressive cytokines, in particular TGF-ß, which quench inflammation and prevent the maturation of antigen-presenting DCs. Tolerance induction is the likely default outcome^{ref1, ref2}. The events recruited by the recognition of exposed PS residues, including the release of immunosuppressive cytokines, possibly contribute to the tolerogenic outcome^ref. However, environmental signals, including signals recruiting the CD40 receptor^ref, delivered by local cytokines, or possibly released by the dying cells themselves^{ref1, ref2}, switch the response toward active cross-priming^ref. Dying tumor cells represent a source of antigens for active antineoplastic immunotherapy. Antigen-presenting DCs efficiently phagocytose dying tumor cells and prime immune responses against tumor antigens both in vitro and, in suitable conditions, in vivo. However, this is not the rule in vivo. Disrupting the local PS-dependent clearance of dying cells by monocytes/macrophages would result in "unscavenged" dead tumor cells, available for uptake and processing by DCs : the PS-binding protein AxV is a suitable tool to obtain this proof of concept because it interferes with the in vitro clearance of dying irradiated tumor cells (ITCs) by monocytes/macrophages, consistent with previous observations^{ref1, ref2, ref3, ref4}. The engulfment of apoptotic cells is a 2-step process. The first involves binding without actual internalization. Ingestion of cell corpses is the second step^ref. Several receptors, including CD14, CD36, and a_v integrins, are involved in the recognition of apoptotic cells ("tethering" receptors). However, the ingestion by macropinocytosis requires recognition of PS on the membranes of the apoptotic cells^ref. AxV efficiently binds to exposed PS. Without the PS "tickle" signal, which is necessary for the reorganization of the actin-based cytoskeleton upon activation of Rho GTPases^ref, monocytes/macrophages internalization fails. PS recognition also elicits the release of immunosuppressive cytokines, namely TGF-ß^ref, and thioglycollate-elicited monocytes/macrophages clearing ITCs in the presence of AxV secreted a significantly lower amount of TGF-ß and higher amounts of IL-1ß and TNF-a. Immature DCs, which are highly macropinocytic and express the constitutively activated Rho GTPase Cdc42, would be less dependent than monocytes/macrophages on tickling signals to internalize tethered apoptotic cells^ref. AxV, which hinders PS-elicited internalization of apoptotic cells, but not their "tethering" via CD36 and the a_v integrin^{ref1, ref2}, did not influence phagocytosis by DCs. Actually, possibly due to the decreased in vivo clearance by elicited monocytes/macrophages, splenic CD11c⁺ DCs phagocytosed more efficiently ITCs injected in the presence of AxV. Of importance, the clearance by DCs both in the presence or in the absence of AxV was restricted in vivo to CD8⁺ DCs. This is an agreement with papers that implicate CD8⁺ DCs in the cross-presentation of cell-associated antigens^{ref1, ref2, ref3, ref4, ref5, ref6}. Accordingly, AxV significantly increased the fraction of CD8⁺ DCs that phagocytose ITCs, but did not influence the phagocytosis by CD8^– DCs. The molecular basis of the different ability of monocytes/macrophages, CD8⁺, and CD8^– DCs to phagocytose dying cells is the object of intense investigation. However, it has been suggested that CD8⁺ DCs selectively express a receptor for apoptotic cells^{ref1, ref2}. Recognition via this yet unidentified moiety is not affected by the 2D crystalline lattice AxV forms on membrane phospholipids. CD8⁺ DCs participate in both cell priming and cell tolerance to cell-associated antigens^{ref1, ref2, ref3, ref4, ref5}. The factors dictating the outcome of cross-presentation include microbial products and endogenous proinflammatory signals, which convert a tolerogenic response by CD8⁺ DCs into productive immunity^ref. Endogenous factors possibly released as a consequence of delayed clearance by scavenger phagocytes are likely to be involved^ref. In agreement, the administration of AxV-coupled ITCs into naive animals protected them from the challenge with living lymphoma cells. The protection achieved was long lasting and specific. AxV-coupled cells were significantly more immunogenic than their untreated counterparts. This number of cells proved highly efficient in causing the rejection of tumors already growing in vivo. The result required the presence of AxV, with statistically significant difference in the latency with which the tumor appeared, in the survival of vaccinated mice and in the number of protected mice. Immunity does not spontaneously initiate when tissue cells die by necrosis and release autoantigens and intracellular adjuvants^ref. This highlights a role for immunoregulatory activities recruited at the site of dying cell clearance. Scavenger monocytes/macrophages releasing immunosuppressive cytokines are possibly involved. Intriguingly, the failure in the function of monocytes/macrophages is involved in the pathogenesis of the prototypic autoimmune disease systemic lupus erythematosus, and the defective clearance of dead cells associates to persistent autoimmunity in vivo^{ref1, ref2, ref3}. Tumor-associated antigens, contained in dying cells, can elicit immune responses. However, they normally fail to do so. Identical constraints are likely to restrict the onset of noxious autoimmune responses and protective tumor-specific immunity^ref. The immunogenicity of dying tumor cells was increased by interfering with the PS-dependent recognition^ref and immunosuppressive clearance of dying cells, a mechanism involved in the physiological maintenance of peripheral tolerance^ref.

One consequence of living in an aerobic environment is an inexorable oxidative modification of molecular targets in vivo. At the cellular level the effects of the oxidized lipid and lipoprotein by-products can include leukocyte recruitment, activation, and apoptosis. Accumulating at sites of inflammation, these oxidation products can have profound pathological consequences, such as in the case of developing arterial lesions^{ref1, ref2, ref3, ref4, ref5}. Formation of oxidized lipids and lipoproteins in inflammation has thus been linked to the advancement of atherosclerosis and other degenerative diseases of aging. Evolution has provided us with various means to cope with oxidative insults and challenges. A growing body of evidence suggests that distinct host defense mechanisms have evolved to dispose of damaged molecular complexes and hopelessly injured cells by means of recognition of oxidized phosphatidylcholine (oxPC) species :

• one such pathway involves recognition of modified lipoproteins and apoptotic or senescent cells by specific scavenger receptors involved in innate immunity, such as CD36 and SR-BI
• an alternative pathway involves recognition of oxPC species and protein-oxPC adducts on lipoproteins through autoantibodies and subsequent Fc-g receptor-mediated endocytosis
• modifications to phosphatidylcholine, the principal phospholipid present in low density lipoprotein (LDL) and cell membranes, render lipoprotein particles and apoptotic cells recognizable by C-reactive protein (CRP)^ref. Under certain conditions, CRP binding may mark or opsonize LDL for macrophage^ref or smooth muscle cell^ref recognition. CRP-mediated enhancement in the uptake of modified forms of LDL could then lead to cholesterol accumulation and formation of foam cells, the characteristic cells of the early atherosclerotic lesions termed "fatty streaks."

• exacerbates the severity of inflammation and the progression of arterial lesions
• reflects attempts by the body to protect itself
• accumulates as an inconsequential epiphenomenon

Molecular interaction map

• click here to view the signal transduction pathways leading to apoptosis.
• overview : regulation of apoptosis
• inhibition of apoptosis
• death receptor signaling
• mitochondrial control of apoptosis
• cellular inhibitor of apoptosis proteins (cIAPs) / Baculoviral IAP repeat-containing (BICR) :
• BICR1 / IAP1
• BICR2 / HIAP2 / IAP2
• BICR3 / HIAP1
• BICR4 / XIAP
• BICR5 / survivin : TCF/ß catenin increases expression of survivin by 6-12-fold in colorectal cancer cells.
• BICR6 / apollon
• BICR7 / livin / ML-IAP / KIAP
• BICR8
• BICR9
• caspases are a family of cysteine proteases (i.e. Cys in the active site) that cleave ⁺H₃N- ... -Asp-Xaa- ...-COO^- bond : procaspases consist of p10 + p20 + NTprodomain.
• CED9 is the Caenorhabditis elegans ortholog for the mammalian antiapoptotic members of the Bcl-2 family.
• Bcl-2 protein is also found in the bilayers of the nuclear envelope and of the endoplasmic reticulum.
• DFF plays the more prominent role in DNA fragmentation and apoptosis in mammals and EndoG likely facilitates DFF function in DNA fragmentation and apoptosis in vivo. EndoG is required for both embryogenesis (through apoptosis at the blastocyst stage, when cavitation occurs that converts a solid embryo into a hollow, 2-layered egg cylinder) and normal apoptosis.
• staurosporine and etoposide trigger mitochondrial-dependent apoptosis in activated peripheral blood lymphocytes.

• loss of phospholipid bilayer asymmetry
• exposure of eat-me flags on the outer leaflet of the plasma membrane which facilitate clearance of apoptotic cell by phagocytes.
• oxidized PtS : this change is absolutely required for recognition  and engulfment to occur. While PtS is actively transported from the outer to the inner leaflet of the plasma membrane by ATP-dependent aminophospholipid translocase, the implication of a scramblase that moves phospholipids bidirectionally across the membrane is still debated. Cyt c released from mitochondria selectively peroxidizes phosphatidylserine (PtS). Peroxidized phosphatidylserine inhibits the ATP-dependent aminophospholipid translocase, allowing oxPtS and phosphatidylethanolamine (PtE) to be exposed also on the extracellular layer of cell membrane. Oxidized PtS is then bound by ...
• surface protein of macrophages ...
• phosphatidylserine receptor
• CD14
• CD36 associated with a_Vb₃ or a_Vb₅ vitronectin receptors
• CD68
• ... and some soluble proteins :
• protein S
• histidine-rich glycoprotein (HRG) is a 75-kDa protein synthetized by mammal hepatocytes and released into the bloodstream at high rates (t_1/2 ~3 days), maintaing a relatively high plasma concentration of 110 ± 25 mg/mL (1.5 ± 0.3 mM) : it acts as a bridge between DNA on apoptotic cells and FcgRI on human monocyte-derived macrophages.
• milk fat globule-EGF-factor-8 (MFG) produced by activated macrophages facilitates recognition by a_Vb₃

Tools for apoptosis detection :

• terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL)
• comet assay / single cell gel electrophoresis (SCGE)
• annexin V assay
• hypodiploid peak at FACS
• staining methods using fluorescence dyes
• 7-aminoactinomycin D (7-AAD) assay can be used to detect the loss of membrane integrity during apoptosis

Web resources :

• Apoptosis MiniCOPE Dictionary
• Apoptosis I
• Apoptosis II
• Apoptosis from Kimball's Biology Pages
• Apoptosis pathways and drug targets poster at Nature Reviews Drug Discovery and Molecular Cell Biology
• Apoptosis glossary
• Apoptosis Online
• Arrestins as signaling molecules involved in apoptotic pathways
• List of apoptosis regulators
• Mitochondrial control of apoptosis in ageing and exercise
• Molecular events regulating active cell death
• The Virtual Library - Apoptosis

Copyright © 2001-2005 Daniele Focosi. All rights reserved  |  Terms of use  | Legal notices
About this site  |  Site map  |  Acknowledgements | Open forum  |  Tell a friend about this site  |  Current link partners
 Abbreviations and acronyms  |  Medical terminology  |  Add a link  |  Translate this site into your favourite language  |  Softwares

---