PML induces a novel caspase-independent death process.

PML nuclear bodies (NBs) are nuclear matrix-associated structures altered by viruses and oncogenes. We show here that PML overexpression induces rapid cell death, independent of de novo transcription and cell cycling. PML death involves cytoplasmic features of apoptosis in the absence of caspase-3 activation, and caspase inhibitors such as zVAD accelerate PML death. zVAD also accelerates interferon (IFN)-induced death, suggesting that PML contributes to IFN-induced apoptosis. The death effector BAX and the cdk inhibitor p27KIP1 are novel NB-associated proteins recruited by PML to these nuclear domains, whereas the acute promyelocytic leukaemia (APL) PML/RAR alpha oncoprotein delocalizes them. Arsenic enhances targeting of PML, BAX and p27KIP1 to NBs and synergizes with PML and IFN to induce cell death. Thus, cell death susceptibility correlates with NB recruitment of NB proteins. These findings reveal a novel cell death pathway that neither requires nor induces caspase-3 activation, and suggest that NBs participate in the control of cell survival.

Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals.

In immature thymocytes, T cell receptor for antigen (TCR) mobilization leads to an active T cell suicide process, apoptosis, which is involved in the selection of the T cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals (Ameisen, J.C., and A. Capron. 1991. Immunol. Today. 4:102). Here, we report that the selective failure of CD4+ T cells from 59 clinically asymptomatic HIV-infected individuals to proliferate in vitro to TCR mobilization by major histocompatibility complex class II-dependent superantigens and to pokeweed mitogen (PWM) is due to an active CD4+ T cell death process, with the biochemical and ultrastructural features of apoptosis. Activation-induced cell death occurred only in the CD4+ T cell population from HIV-infected asymptomatic individuals and was not observed in T cells from any of 58 HIV-seronegative controls, including nine patients with other acute or chronic infectious diseases. Activation-induced CD4+ T cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody (mAb). The CD28 mAb not only prevented apoptosis but also restored T cell proliferation to stimuli, including PWM, superantigens, and the tetanus and influenza recall antigens. These findings may have implications for the understanding of the pathogenesis of acquired immune deficiency syndrome and for the design of specific therapeutic strategies.

T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons.

Human immunodeficiency virus (HIV) infection leads to a progressive loss of CD4+ T helper (Th) type 1 cell-mediated immunity that is associated with defective in vitro CD4+ T cell proliferation and abnormal T cell death by apoptosis in response to T cell receptor (TCR) stimulation. Quantification of interleukin (IL)-2, interferon gamma, IL-4, IL-5, and IL-10 secretion by immunoassays, and of interferon gamma, IL-4 and IL-10 messenger RNA expression by competitive reverse transcriptase polymerase chain reaction after in vitro stimulation of the TCR revealed a similar Th1 cytokine profile in T cells from HIV-infected persons and from controls. These data indicated that the loss of CD4+ Th1 cell function in HIV-infected persons is not related to a Th1 to Th2 cytokine switch as previously proposed, but to a process of activation-induced death of CD4+ Th1 cells. Despite the absence of elevated levels of Th2 cytokines, apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented in vitro by antibodies to IL-10 or IL-4, two Th2 cytokines that downregulate Th1 cell responses, or by the addition of recombinant IL-12, a cytokine that upregulates Th1 functions. TCR-induced apoptosis of T cell hybridomas and preactivated T cells has been shown to involve the CD95 (Fas/Apo-1) molecule. CD4+ and CD8+ T cells from HIV-infected persons expressed high levels of the CD95 molecule, and, in contrast to T cells from controls, were highly sensitive to antibody-mediated CD95 ligation, which induced apoptosis in a percentage of T cells similar to that induced by TCR stimulation. As TCR-induced apoptosis, CD95-mediated apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented by the addition of recombinant IL-12. Together, these findings suggest that apoptosis of CD4+ T cells from HIV-infected persons involves an abnormal sensitivity to CD95 ligation, and to TCR stimulation in the presence of normal levels of Th2 cytokines. The preventive effect of IL-12 on both mechanisms has potential implications for the design of immunotherapy strategies aimed at the upregulation of CD4+ Th1 cell functions in AIDS.

Immunological crossreactivity between the human immunodeficiency virus type 1 virion infectivity factor and a 170-kD surface antigen of Schistosoma mansoni.

A monoclonal antibody (mAb) directed against a synthetic peptide derived from the sequence of the human immunodeficiency virus type 1 (HIV-1) regulatory protein virion infectivity factor (vif) labeled the surface of Schistosoma mansoni schistosomula by indirect immunofluorescence. Western blotting showed that two S. mansoni proteins of 170 and 65 kD were recognized by the mAb. Sera from 20% of S. mansoni-infected HIV-seronegative individuals tested recognized the PS4 peptide in an ELISA as did sera from S. mansoni-infected rats. Sera from individuals seropositive for HIV-1, but without schistosomiasis, that reacted with the vif peptide also recognized a 170-kD S. mansoni protein. This crossreactive S. mansoni antigen appears to be a target of immunity in vivo since passive transfer of the mAb VIF-CD3 to naive rats had a protective effect against a challenge infection with S. mansoni cercariae.

Functional study of a monoclonal antibody to IgE Fc receptor (Fc epsilon R2) of eosinophils, platelets, and macrophages.

An IgM mAb (BB10) was produced by immunization of mice with human eosinophils purified according to their abnormal low density ("hypodense" cells), and previously shown to exhibit increased IgE-dependent antiparasite cytotoxicity. This BB10 antibody, selected for positive fluorescence staining of hypodense blood or lung eosinophils and low or negative staining of normodense eosinophils or neutrophils, could strongly inhibit IgE-dependent cytotoxicity of human eosinophils and platelets. The specificity for the IgE Fc receptor was suggested by the high levels of inhibition of IgE rosettes formed by eosinophils after incubation with the purified IgM fraction of BB10, whereas other receptors (Fc gamma R, CR1) were not affected. On the other hand, BB10, able to inhibit rat eosinophil Fc epsilon R, did not react with the IgE Fc receptor on mast cells or basophils. A technique using radioiodinated BB10 allowed us to quantify the specific binding of BB10 to human eosinophils and platelets. Competition experiments revealed a crossinhibition between the binding of BB10 and IgE, suggesting the specificity of BB10 for the IgE binding site of eosinophil, platelet, and monocyte Fc epsilon R. Three proteins having extrapolated Mr of 32,000, 43,000-45,000, and 97,000 were found in the platelet extract eluted from a BB10 or from an IgE immunosorbent column. These findings confirm the similarities between IgE Fc receptors on human eosinophils, platelets, and macrophages, already observed with polyclonal antibodies directed against the B lymphocyte Fc epsilon receptor. They suggest, moreover, that the mAb BB10 can represent a good reagent for further investigations on the structure and the functions of this IgE Fc receptor (Fc epsilon R2).

The relevance of apoptosis to AIDS pathogenesis.

Several recent experimental findings support the hypothesis that apoptosis induced by human immune deficiency virus (HIV) is important in the pathogenesis of acquired immune deficiency syndrome (AIDS). Thus, one potential therapeutic strategy against AIDS may be to block the HIV-mediated apoptosis signal transduction pathway. Induction of apoptosis by HIV infection may prove a useful paradigm for the pathogenesis of a wide range of diseases that involve cell depletion and tissue atrophy.

Endothelial cell apoptosis in severe drug-induced bullous eruptions.

BACKGROUND: Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are characterized by extensive keratinocyte apoptosis mediated by cytotoxic proteins. Similar features have been found in another severe dysimmune syndrome, allogeneic acute graft-versus-host disease, where endothelial cell apoptosis has been recently characterized. OBJECTIVES: To determine whether endothelial cell apoptosis occurs in dermal vessels of TEN and SJS, and whether it is linked to expression of cytotoxic proteins. METHODS: Skin biopsies of eight patients with severe drug-induced bullous eruptions (four TEN, four SJS), eight with drug-induced urticaria and eight healthy controls were compared. Blood vessel damage was studied by electron microscopy and quantified by CD31 immunostaining. Apoptotic cells, characterized by electron microscopy, were quantified on terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay. Immunohistochemistry was also used to characterize and quantify inflammatory cells and granzyme B, tumour necrosis factor (TNF)-alpha and Fas ligand (FasL) expression. RESULTS: Endothelial cell apoptosis was observed in all TEN and SJS cases: it occurred in 85% of the vessel sections. It occurred in one case of drug-induced urticaria, in 5% of vessel sections, but not in healthy controls. Numbers of CD68+ macrophages and CD8+ T lymphocytes were significantly higher in TEN and SJS compared with both other groups; granzyme B and TNF-alpha but not FasL were expressed. CONCLUSIONS: Characterization of endothelial cell apoptosis in TEN and SJS is important to assess a factor worsening skin damage, with possible extension to other organs. It may also be useful for the development of novel therapeutic strategies.

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death.

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.

On the origin, evolution, and nature of programmed cell death: a timeline of four billion years.

Programmed cell death is a genetically regulated process of cell suicide that is central to the development, homeostasis and integrity of multicellular organisms. Conversely, the dysregulation of mechanisms controlling cell suicide plays a role in the pathogenesis of a wide range of diseases. While great progress has been achieved in the unveiling of the molecular mechanisms of programmed cell death, a new level of complexity, with important therapeutic implications, has begun to emerge, suggesting (i) that several different self-destruction pathways may exist and operate in parallel in our cells, and (ii) that molecular effectors of cell suicide may also perform other functions unrelated to cell death induction and crucial to cell survival. In this review, I will argue that this new level of complexity, implying that there may be no such thing as a 'bona fide' genetic death program in our cells, might be better understood when considered in an evolutionary context. And a new view of the regulated cell suicide pathways emerges when one attempts to ask the question of when and how they may have become selected during evolution, at the level of ancestral single-celled organisms.

Apoptosis in a unicellular eukaryote (Trypanosoma cruzi): implications for the evolutionary origin and role of programmed cell death in the control of cell proliferation, differentiation and survival.

The origin of programmed cell death (PCD) has been linked to the emergence of multicellular organisms. Trypanosoma cruzi, a member of one of the earliest diverging eukaryotes, is a protozoan unicellular parasite that undergoes three major differentiation changes and requires two different hosts. We report that the in vitro differentiation of the proliferating epimastigote stage into the G0/G1 arrested trypomastigote stage is associated with massive epimastigote death that shows the cytoplasmic and nuclear morphological features and DNA fragmentation pattern of apoptosis, the most frequent phenotype of PCD in multicellular organisms. Apoptosis could be accelerated or prevented by modifying culture conditions or cell density, indicating that extracellular signals influenced the epimastigote decision between life and death. Epimastigotes responded to complement-mediated immunological agression by undergoing apoptosis, while undergoing necrosis in response to nonphysiological saponin-mediated damage. PCD may participate into the optimal adaptation of T. cruzi to its different hosts, and the avoidance of a local competition between a G0/G1 arrested stage and its proliferating progenitor. The existence of a regulated cell death programme inducing an apoptotic phenotype in a unicellular eukaryote provides a paradigm for a widespread role for PCD in the control of cell survival, which extends beyond the evolutionary constraints that may be specific to multicellular organisms and raises the question of the origin and nature of the genes involved. Another implication is that PCD induction could represent a target for therapeutic strategies against unicellular pathogens.

A novel mechanism for HIV1-mediated bystander CD4+ T-cell death: neighboring dying cells drive the capacity of HIV1 to kill noncycling primary CD4+ T cells.

CD4+ T-cell death is a crucial feature of AIDS pathogenesis, but the mechanisms involved remain unclear. Here, we present in vitro findings that identify a novel process of HIV1 mediated killing of bystander CD4+ T cells, which does not require productive infection of these cells but depends on the presence of neighboring dying cells. X4-tropic HIV1 strains, which use CD4 and CXCR4 as receptors for cell entry, caused death of unstimulated noncycling primary CD4+ T cells only if the viruses were produced by dying, productively infected T cells, but not by living, chronically infected T cells or by living HIV1-transfected HeLa cells. Inducing cell death in HIV1-transfected HeLa cells was sufficient to obtain viruses that caused CD4+ T-cell death. The addition of supernatants from dying control cells, including primary T cells, allowed viruses produced by living HIV1-transfected cells to cause CD4+ T-cell death. CD4+ T-cell killing required HIV1 fusion and/or entry into these cells, but neither HIV1 envelope-mediated CD4 or CXCR4 signaling nor the presence of the HIV1 Nef protein in the viral particles. Supernatants from dying control cells contained CD95 ligand (CD95L), and antibody-mediated neutralization of CD95L prevented these supernatants from complementing HIV1 in inducing CD4+ T-cell death. Our in vitro findings suggest that the very extent of cell death induced in vivo during HIV1 infection by either virus cytopathic effects or immune activation may by itself provide an amplification loop in AIDS pathogenesis. More generally, they provide a paradigm for pathogen-mediated killing processes in which the extent of cell death occurring in the microenvironment might drive the capacity of the pathogen to induce further cell death.

On the evolution of programmed cell death: apoptosis of the unicellular eukaryote Leishmania major involves cysteine proteinase activation and mitochondrion permeabilization.

Leishmania major is a protozoan parasite from one of the most ancient phylogenic branches of unicellular eukaryotes, and containing only one giant mitochondrion. Here we report that staurosporine, that induces apoptosis in all mammalian nucleated cells, also induces in L. major a death process with several cytoplasmic and nuclear features of apoptosis, including cell shrinkage, phosphatidyl serine exposure, maintenance of plasma membrane integrity, mitochondrial transmembrane potential (DeltaPsim) loss and cytochrome c release, nuclear chromatin condensation and fragmentation, and DNA degradation. Nuclear apoptosis-like features were prevented by cysteine proteinase inhibitors, and cell free assays using dying L. major cytoplasmic extracts indicated that the cysteine proteinases involved (i) also induced nuclear apoptosis-like features in isolated mammalian nuclei, and (ii) shared at least two nuclear substrates, but no cleavage site preference, with human effector caspases. Finally, isolated L. major mitochondria released cytochrome c and cysteine proteinases with nuclear pro-apoptotic activity when incubated with human recombinant Bax, even (although much less efficiently) when Bax was deleted of its transmembrane domain required for insertion in mitochondrial outermembranes, implying that L. major mitochondrion may express proteins able to interact with Bax. The recruitment of cysteine proteinases and mitochondria to the cell death machinery may be of very ancient evolutionary origin. Alternately, host/parasite interactions may have exerted selective pressures on the cell death phenotype of kinetoplastid parasites, resulting in the more recent emergence of an apoptotic machinery through a process of convergent evolution.

Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria.

Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.

Caspase-dependent and -independent T-cell death pathways in pathogenic simian immunodeficiency virus infection: relationship to disease progression.

Studies of human immunodeficiency virus (HIV) and nonhuman primate models of pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections have suggested that enhanced ex vivo CD4 T-cell death is a feature of pathogenic infection in vivo. However, the relative contributions of the extrinsic and intrinsic pathways to programmed T-cell death in SIV infection have not been studied. We report here that the spontaneous death rate of CD4+ T cells from pathogenic SIVmac251-infected rhesus macaques ex vivo is correlated with CD4 T-cell depletion and plasma viral load in vivo. CD4+ T cells from SIVmac251-infected macaques showed upregulation of the death ligand (CD95L) and of the proapoptotic proteins Bim and Bak, but not of Bax. Both CD4+ and CD8+ T cells from SIVmac251-infected macaques underwent caspase-dependent death following CD95 ligation. The spontaneous death of CD4+ and CD8+ T cells was not prevented by a decoy CD95 receptor or by a broad-spectrum caspase inhibitor (zVAD-fmk), suggesting that this form of cell death is independent of CD95/CD95L interaction and caspase activation. IL-2 and IL-15 prevented the spontaneous death of CD4+ and CD8+ T cells, whereas IL-10 prevented only CD8 T-cell death and IL-7 had no effect on T-cell death. Our results indicate that caspase-dependent and caspase-independent pathways are involved in the death of T cells in pathogenic SIVmac251-infected primates.

T helper cell epitopes of the human immunodeficiency virus (HIV-1) nef protein in rats and chimpanzees.

T helper cell antigenic and immunogenic determinants of the nef protein were investigated in the rat and chimpanzee models using recombinant nef protein and five synthetic peptides selected according to their amphipathic and alpha-helicity properties. The nef protein was shown to be immunogenic with both Freund's or aluminium hydroxide adjuvants. After immunization with the nef protein the 45-69 peptide was the most antigenic in rat and monkey models. In contrast, the 98-112 peptide, that required a carrier protein to induce in vitro rat T cell recall proliferation, was able to restimulate monkey T cells in the absence of a carrier. The amino acid sequence carrying the antigenic activity of the 45-69 peptide was further investigated by synthesizing short peptides overlapping this region. The antigenic sequence was precisely located in the middle of the peptide (region 50-59). This sequence was antigenic only when N alpha-acetylated. Circular dichroism analysis of the 45-69 peptide and the in vitro activity of the N-terminus group indicate in this case the involvement of the alpha-helical propensity for antigen presentation. However, the shorter sequence 50-64, able to induce a T cell reactivity, was determined as a beta-pleated sheet structure in aqueous solution. The 45-69 peptide was not only antigenic but also immunogenic and behaved in vivo as a functional T helper cell epitope. Indeed, the priming with the peptide or the transfer of peptide specific T cells to a naive recipient, followed by immunization with the nef protein, enhanced the subsequent antibody response to the nef protein. Together, these data indicate that the 45-69 peptide appears as a candidate for the in vivo elicitation of T cell immunity to the HIV-1 nef regulatory protein.

Determination of B-cell epitopes of nef HIV-I protein: immunogenicity related to their structure.

Determination of the B-cell epitopes of the nef molecule encoded by the human immunodeficiency virus type 1 (HIV-1) was undertaken using a set of six synthetic peptides. Sequences that were both antigenic and immunogenic and stimulated the production of antibodies recognizing the full length molecule, were considered as B-cell epitopes. Two peptidic sequences were antigenic both in rodents (mice and rats) and in non-human primates (chimpanzee). They were located in the regions 45-69 and 176-206 of the nef molecule. Two additional antigenic sequences were determined, one in chimpanzees, region 79-94, and the second in rodents, region 148-161. Immunogenicity was investigated in the rodents. Only the 45-69 and 176-206 sequences were immunogenic, and specific antibodies present in the sera of the immunized animals reacted with the nef protein. Therefore, each of these two sequences could be considered as containing at least one B-cell epitope. The fine epitopic specificity was determined using subfragments of these two sequences and it was shown that the antigenic determinants were contained in the C-terminal region of each sequence overlapping with the T-cell epitopes. These results raised the importance of vicinity of B- and T-cell determinants and their immunogenicity.

Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis.

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.

Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review.

Human red blood cells (RBCs) have a life-span of 120 days in circulation, after which they are phagocytized by resident macrophages. Extensive studies have been undertaken by many investigators in order to elucidate the cellular and molecular mechanisms of the erythrophagocytosis. The critical questions addressed by physiologists, clinicians and biochemists are: 'which of the many traumatic blemishes that appear on the erythrocyte surface as it winds its way through the circulation is the primary signal for clearance of the effete RBC from the circulation?', or 'What is the critical signal that it, and it alone, will activate the resident macrophage to adhere to and engulf it?'. Numerous, and often conflicting, hypotheses have been proposed. Each investigator focusing on but one of the many modifications that afflict the cell surface of the ageing erythrocyte, viz changes in either or both the carbohydrate or peptidic moieties of glycoproteins; abolishment of the pre-existing asymmetry in the lipid bilayer with the exposure of phosphatidylserine residues; or alterations in spectrin, to mention but a few. Many of these investigators also have invoked an intermediary role for auto-immune antibodies that recognise the change(s) on the erythrocyte surface and thereby serve as opsonins as a prelude to the erythrophagocytosis. The objective of the present review is to evaluate the data in support of the various hypotheses, and to submit some of our own recent observations involving the use of flow cytometric procedures that: i) provide evidence that the cell surface sialic acid serves as a determinant of the life-span; ii) characterise the senescent erythrocyte population that is specifically captured and phagocytized by macrophages (utilising the rapid and sensitive procedure we developed for quantification of in vitro erythrophagocytosis); and finally iii) provide evidence for the existence of an alternative pathway that is independent of immunoglobulins.

YOPRO-1 permits cytofluorometric analysis of programmed cell death (apoptosis) without interfering with cell viability.

In the absence of cell permeabilization, the impermeant nuclear dye YOPRO-1 permits accurate analysis of apoptosis using cytofluorometry or fluorescent microscopy. Several immune cell populations were studied including dexamethasone-treated thymocytes, irradiated peripheral blood mononuclear cells and a growth factor-depleted tumor B cell line. Excellent correlation values were found with acridine orange using cytofluorometry and with eosin-hematoxylin using optical microscopy. Under fluorescent microscopy, YOPRO-1-fluorescent cells demonstrate the morphological features of cells undergoing apoptosis such as nuclear shrinkage and fragmentation. An important characteristic of the dye that differs from all other nuclear dyes previously used for the detection of apoptosis is that it does not label living cells. Cell sorting after flow cytofluorometry analysis confirmed that only the apoptotic cell population was labelled with YOPRO-1. Further studies showed that while incubation of living cells with Hoechst 33342 almost completely abrogated the capacity of T cells to proliferate in response to several stimuli, YOPRO-1 had no inhibitory effect. This new simple, rapid and reproducible use of the YOPRO-1 dye should prove useful in the analysis of apoptotic cells as well as for investigations of the functional properties of living cells in a culture containing apoptotic cells.