Apoptotic DNA alterations in pig leukocytes after phagocytosis of bacteria are linked to maturation of the immune system

The effect of phagocytosis of living bacteria on apoptotic DNA changes was examined in pig leukocytes in relation to immune system maturation. Blood samples of pigs (aged 6, 12 and 18 weeks) were cultivated with a suspension of bacterial cells Salmonella typhimurium LB 5000 at 37 (o)C. In the experimental groups, killed bacteria and microspheric particles were used to detect the influence of the phagocytic process. Phagocytic activity and index were determined in each sample by means of microspheric particles. The ability to kill engulfed microbes (bactericidal capacity) was estimated from the decrease in bacterial colony-forming units (CFU). Samples of cultured cells were taken for DNA analysis at given intervals. DNA ladder assay was used for qualitative apoptotic DNA break detection and the TUNEL AP test was employed for quantification of apoptosis. In 18-week-old animals, spontaneous DNA degradation was observed in the control group without phagocytosis after 8 h. In contrast, cells cultivated with microspheric particles or killed bacteria became apoptotic after 4 h. The rate of apoptotic DNA degradation was decreased in the group exposed to living bacteria. This prolonged survival of phagocytes was also detected in 12-week-old animals, but not at 6 weeks of age. These findings were supported by the ability of phagocytes in 6-week-old animals to engulf microbes, but their killing (bactericidal) ability was significantly decreased in comparison with other stages of immune system maturation. These results suggest that the process of phagocytosis itself is accompanied by activation of the apoptotic program in phagocytic cells of the pig immune system, but the presence of phagocyted living bacteria can delay this activation. The prolonged survival of short-lived cells was only observed in later phases of immune system maturation.     

Effect of Bacillus anthracis lethal toxin on human peripheral blood mononuclear cells

Lethal toxin (LeTx) plays a central role in anthrax pathogenesis, however a cytotoxicity of LeTx has been difficult to demonstrate in vitro. No cytolytic effect has been reported for human cells, in contrast to murine cell lines, indicating that cell lysis can not be considered as a marker of LeTx activity. We have recently shown that murine macrophage-like RAW 264.7 cells treated with LeTx or infected with anthrax spores underwent changes typical of apoptotic death. Here we demonstrate that cells from human peripheral blood display a proapoptotic behavior similar to murine cells. TUNEL assay detected a nucleosomal degradation typical of apoptosis in peripheral blood mononuclear cells (PBMC) treated with LeTx. Membrane staining with apoptotic dyes was detected in macrophages derived from monocytes in presence of LeTx. The toxin inhibited production of proinflammatory cytokines in PBMC stimulated with a preparation of Bacillus anthracis cell wall. Infection of PBMC with anthrax spores led to the appearance of a large population of cells stained positively for apoptosis, with a reduced capacity to eliminate spores and vegetative bacteria. The aminopeptidase inhibitor, bestatin, capable of protecting cells from LeTx, restored a bactericidal activity of infected cells. These findings may be explained by LeTx expression within phagocytes and support an important role of LeTx as an early intracellular virulence factor contributing to bacterial dissemination and disease progression.     

Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin

The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked β-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.     

Caspase Activation Is an Early Event in Anthracycline-Induced Apoptosis and Allows Detection of Apoptotic Cells before They Are Ingested by Phagocytes

An increasing number of methods are being described to detect apoptotic cells. However, attempts to detect apoptotic cells in clinical samples are rarely successful. A hypothesis is that apoptotic cells are cleared from the circulation by phagocytosis before they become detectable by conventional morphological or cytometric methods. Using LR73 adhering cells as phagocytes in a model ofin vitrophagocytosis, we found that phagocytosis of daunorubicin (DNR)-treated U937, HL60, or K562 leukemia cell lines occurred prior to phosphatidylserine externalization, DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mitochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis assay to detect caspase activity in ceramide- or DNR-treated cells (fluorogenic substrate for caspase), we found that caspase activity increased in apoptosis-committed cells before they were detected by flow cytometry or recognized by phagocytes. Similarly a caspase activity increase was detected in circulating mononuclear cells of leukemic patients 15 h after the beginning of anthracyclin treatment. We suggest that recent findings on enzymatic events (caspase activation) occurring in the early events of apoptosis must now allow the development of new markers for apoptosis, irrespective of the morphological features or internucleosomal fragmentation which are late events in apoptosis.     

Early ontogeny of monocytes and macrophages in the pig

Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.     

Apoptotic cells induce a phosphatidylserine-dependent homeostatic response from phagocytes

Engulfment of apoptotic cells by phagocytes is important throughout development and adult life. When phagocytes engulf apoptotic cells, they increase their cellular contents including cholesterol and phospholipids, but how the phagocytes respond to this increased load is poorly understood. Here, we identify one type of a phagocyte response, wherein the recognition of apoptotic cells triggers enhanced cholesterol efflux (to apolipoprotein A-I) from macrophages. Phosphatidylserine (PS) exposed on apoptotic cells was necessary and sufficient to stimulate the efflux response. A major mechanism for this enhanced efflux by macrophages was the upregulation of the mRNA and protein for ABCA1, a membrane transporter independently linked to cholesterol efflux as well as engulfment of apoptotic cells. This increase in phagocyte ABCA1 levels required the function of nuclear receptor LXRalpha/beta, a known regulator of cholesterol homeostasis in humans and mice. Taken together, these data reveal a "homeostatic program" initiated in phagocytes that include a proximal membrane signaling event initiated by PS recognition, a downstream signaling event acting through nuclear receptors, and an effector arm involving upregulation of ABCA1, in turn promoting reverse cholesterol transport from the phagocytes. These data also have implications for macrophage handling of contents derived from apoptotic versus necrotic cells in atherosclerotic lesions.     

Evaluation of the interactions between strains of S. aureus and peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal evaluation these cells were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate the efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocynate (FITC) and the percentage of cells containing FITC-labelled bacteria were analysed by flow cytometry. The data obtained show the strains of S. aureus derived from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from the respiratory tract have the lowest sensitivity to killing. These strains differ too in their ability to trigger monocyte CL response. On the contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed a significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

Evaluation of interactions between strains of S. aureus isolated from different clinical specimens with peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal elutriation were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocyanate (FITC) and the percentage of cells containing FITC-labelled bacteria was analysed by flow cytometry. The data obtained show that strains of S. aureus originated from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from respiratory tract show the lowest sensitivity for killing. These strains differ too in their ability to trigger monocyte CL response. Contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells

A key feature of the process of programmed cell death (apoptosis) is the efficiency with which the dying cells are recognized and engulfed by phagocytes [1]. Apoptotic cells are rapidly cleared either by neighbouring cells acting as semi-professional phagocytes or by experts of the macrophage line, so that an inflammatory response is avoided [2]. The Caenorhabditis elegans gene ced-6 is required for efficient engulfment of apoptotic cells [3] and is one of a group of genes that define two partially redundant parallel pathways for the engulfment process [4] [5]. These pathways may be conserved across evolution, as two other engulfment genes have human homologues. A CED-5 homologue is part of a human CrkII-DOCK180-Rac signaling pathway proposed to mediate cytoskeletal reorganization [6] [7] [8] and a CED-7 homologue is similar to the ABC transporters [9] [10]. Here, we report the cloning and characterization of human CED-6, a human homologue of C. elegans CED-6. The 34 kDa hCED-6 protein is expressed in most tissues, some human cancer cells, and in primary human macrophages. We developed an assay that quantitates the phagocytic activity of mammalian macrophages: the number of apoptotic cells that have been internalized is measured by the uptake of lacZ-positive apoptotic cells by adherent transgenic macrophages. The results of this assay demonstrate that overexpression of hCED-6 promotes phagocytosis only of apoptotic cells and suggest that hCED-6 is the mammalian orthologue of C. elegans CED-6 and is a part of a highly conserved pathway that specifically mediates the phagocytosis of apoptotic cells.     

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.     

Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila

Apoptotic cell phagocytosis is initiated through the specific interaction between markers for phagocytosis present at the surface of targets and their receptors of phagocytes. Although many molecules have been proposed to be phagocytosis markers and receptors in mammals, information as to the identity of those molecules is limited for invertebrate animals. Calreticulin, a molecular chaperone that functions in the lumen of the endoplasmic reticulum, was recently reported to be the second general marker, the membrane phospholipid phosphatidylserine being the first, for mammalian apoptotic cells to be recognized by phagocytes. We here asked whether or not calreticulin serves as a marker for phagocytosis in Drosophila. Phagocytosis of apoptotic S2 cells by Drosophila hemocyte-derived l(2)mbn cells, which we previously showed to occur independent of phosphatidylserine, was inhibited by the addition of anti-calreticulin antibody. This inhibition was observed when the target cells, but not phagocytes, were pre-incubated with the antibody. In addition, RNA interference-mediated reduction of calreticulin expression in apoptotic S2 cells, but not in l(2)mbn cells, reduced the level of phagocytosis. An immunocytochemical analysis revealed that calreticulin is widely distributed at the surface of viable S2 cells. After the induction of apoptosis, cell surface calreticulin seemed to form aggregates, with no change in its amount. Furthermore, in embryos of a mutant Drosophila strain that expresses calreticulin at a reduced level, the level of phagocytosis of apoptotic cells was about a half of that observed in embryos of a wild-type strain. These results collectively indicate that calreticulin is the first molecule to be identified as a marker for phagocytosis of apoptotic cells by Drosophila phagocytes.     

Phagocytosis of apoptotic cells and the resolution of inflammation

Clearance of apoptotic cells by phagocytic cells plays a significant role in the resolution of inflammation, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. Apoptosis induces cell surface changes that are important for recognition and engulfment of cells by phagocytes. These changes include alterations in surface sugars, externalization of phosphatidylserine and qualitative changes in the adhesion molecule ICAM-3. Several studies have contributed to clarify the role of the receptors on the surface of phagocytes that are involved in apoptotic cell clearance. The phagocytic removal of apoptotic cells does not elicit pro-inflammatory responses; in contrast, apoptotic cell engulfment appears to activate signals that suppress release of pro-inflammatory cytokines. Therefore, clearance of apoptotic leucocytes is implicated in the resolution of inflammation and mounting evidence suggests that defective clearance of apoptotic cells contributes to inflammatory and autoimmune diseases. Defining the ligands on apoptotic cells and the corresponding receptors on phagocytes with which they engage, is likely to lead to the development of novel anti-inflammatory pro-resolution drugs. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. We will also discuss what is currently known about diseases that may reflect impaired phagocytosis and the consequences on inflammation and immune responses.     

TNF-alpha coupled to membrane of apoptotic cells favors the cross-priming to melanoma antigens

The cross-presentation of Ags derived from apoptotic cell processing contributes to peripheral tolerance. Environmental signals possibly modify this default outcome, favoring cross-priming. In this study, we anchored via a biotin-avidin-biotin bridge soluble TNF-alpha to the membrane of apoptotic melanoma cells and studied in vivo and in vitro the interaction with Ag-presenting phagocytes. TNF-alpha-coated apoptotic melanoma cells injected s.c. had a faster and more efficient access to draining lymph nodes, with cross-priming of melanoma-specific CTLs and delayed outgrowth of melanomas in all treated animals. Twenty percent of the animals, in the absence of further adjuvant, did not develop the tumor. Immature dendritic cells challenged with TNF-alpha-coated apoptotic melanoma cells secreted proinflammatory cytokines in an autocrine/paracrine fashion, efficiently matured, as assessed functionally and by flow cytometry and cross-presented with enhanced efficiency melanoma Ags to MHC class I- and II-restricted T cells. The results indicate that TNF-alpha targeted to apoptotic membranes, at concentrations that can be safely reached in growing tumors without undue systemic toxicity, influences the outcome of the disposal of dying cells and enhances tumor immunogenicity.     

Cholesterol-induced apoptotic macrophages elicit an inflammatory response in phagocytes, which is partially attenuated by the Mer receptor

Macrophage apoptosis and the ability of phagocytes to clear these apoptotic cells are important processes in advanced atherosclerosis. Phagocytic clearance not only disposes of dead cells but usually elicits an anti-inflammatory response. To study this process in a model of advanced lesional macrophage death, macrophages rendered apoptotic by free cholesterol loading (FC-AMs) were incubated briefly with fresh macrophages ("phagocytes"). FC-AMs were promptly ingested by the phagocytes, which was dependent upon actin polymerization and the phagocyte Mer receptor. Surprisingly, this brief exposure to FC-AMs triggered a modest proinflammatory response in the phagocytes: tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta were induced, whereas the levels of transforming growth factor-beta and IL-10 were not increased. This response required cell contact between the FC-AMs and phagocytes but not FC-AM ingestion. TNF-alpha and IL-1beta induction required one or more proteins on the FC-AM surface and was dependent on signaling through extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase and nuclear factor-kappaB in the phagocytes. TNF-alpha production was markedly greater when Mer-defective phagocytes were used, indicating that Mer attenuated the inflammatory response. Interestingly, a more typical anti-inflammatory response was elicited when phagocytes were exposed to macrophages rendered apoptotic by oxidized low density lipoprotein or UV radiation. Thus, the proinflammatory milieu of advanced atherosclerotic lesions may be promoted, or at least not dampened, by contact between FC-induced apoptotic macrophages and neighboring phagocytes prior to apoptotic cell ingestion.     

Granular biogenic amine-containing cells of the rat endometrium as components of the system of mononuclear phagocytes

To 10 non-inbred white mature rats 24 h before sacrifice 7.0 ml of Indian ink colloid solution has been injected intraperitoneally. Histochemical reactions of Falk-Hillarp (catecholamines, serotonin) have been performed on nonfixed cryostat slices of the uterus, those of Cross, Even, Rost (histamine) against non-specific esterase and acid phosphatase. Before carrying out these reactions, the slices have been examined under luminescent microscope LUMAM-I3 by means of luminescent and usual illumination in order to reveal cells, possessing autoluminiscence and containing phagocyted Indian ink particles. Presence of autoluminescence, phagocyted material, catecholamines and serotonin, histamine, nonspecific esterase, acid phosphotase, prostaglandin E2 in the same granular cells of the rat endometrium have been stated. All cellular properties revealed are specific for macrophages. A conclusion is made that granular biogenic amine-containing cells of endometrium can be considered as belonging to the system of mononuclear phagocytes.     

Contribution of automated hematology analysis to the detection of apoptosis in peripheral blood lymphocytes

Automated hematology analyzers (analyzers) can provide complete blood counts and white blood cell (WBC) differentials in clinical laboratories and alert users to the presence of quantitative and qualitative cell abnormalities through cautionary flags. In this study, we applied analyzers to the screening of apoptotic cells in peripheral blood and examined the triggering capacity of cautionary flags to detect apoptotic cell populations. EDTA-anticoagulated fresh peripheral blood from patients with acute infectious mononucleosis containing atypical lymphocytes comprising 12.3 +/- 4. 0% of WBC was applied to a Beckman-Coulter MAXM A/L Retic (MAXM) analyzer. The lymphocyte cluster spread upward in VOLUME/DF1 scattergrams and the threshold lines between lymphocyte and monocyte clusters shifted upward. Flags for the number and percentage of lymphocytes, variant lymphocytes, and blast cells were generally present for samples containing atypical lymphocytes. After the blood from acute infectious mononucleosis patients was incubated for 4 h at 37 degrees C, peripheral blood smears revealed the presence of morphologically apoptotic cells comprising 9.0 +/- 4.2% of WBC and a comparable reduction of lymphocytes. On the MAXM analyzer, the apoptotic lymphocyte cluster appeared under the lymphocyte cluster in VOLUME/DF1 scattergrams. However, no specific flag was present to alert users to the presence of the apoptotic lymphocyte cluster. We conclude that visual inspection of scattergrams generated by the MAXM analyzer can be useful for the detection of apoptotic lymphocytes in peripheral blood. Cytometry (Comm. Clin. Cytometry) 42:209-214, 2000. Published 2000 Wiley-Liss, Inc.     

A Peptide of CD14 Protects Human Lymphocytes from Gliotoxin-Induced Apoptosis

CD14 is a 56?Kd glycoprotein typically present on myeloid cells and originally implicated in innate immunity and pattern recognition. The possible involvement of its membrane bound form with the clearance of apoptotic cells and its recently demonstrated expression by non-myeloid cells, make this fascinating molecule a target for more research and possible therapeutic use. The objective of this study was to assess the possible anti apoptotic effect of the soluble form of CD14, on human lymphocytes and to delineate the fragment, from within the protein that may be mediating that activity. Human peripheral blood lymphocytes were cultured in vitro and induced for apoptosis with gliotoxin, with or without the addition of rhCD14 or CD14 peptide. As controls, we used lymphocyte cell lines such as Jurkat, NK-YTS and also normal peripheral blood neutrophils. We demonstrate that human soluble CD14 is capable of protecting human lymphocytes from gliotoxin- induced apoptosis in vitro. It is not capable of protecting gliotoxin-treated apoptotic Jurkat or NK-YTS cells. Moreover, we reveal that a fragment of the protein, a peptide that is recognized by the MO2 monoclonal anti-CD14 antibody, retains this protective effect, although with a lower efficiency. We have previously shown that the MO2 epitope was found naturally inside human lymphocytes. Here, we demonstrate that the MO2 peptide can penetrate lymphocytes in human peripheral blood mononuclear cells incubated in vitro. The peptide does not penetrate Jurkat or NK-YTS cells and it does not protect them from apoptosis. In addition, peripheral blood neutrophils, in which the peptide penetration was much better than in lymphocytes, could not be protected by this peptide from their spontaneous apoptosis in vitro.Our data thus suggest that circulating CD14 may play an important role in the prevention of apoptosis of lymphocytes and that a specific region from within the molecule is involved in this activity.     

Cues for apoptotic cell engulfment: eat-me, don't eat-me and come-get-me signals

Efficient elimination of cells undergoing programmed cell death is crucial for normal tissue homeostasis and for the regulation of immune responses. This review examines unique signals presented by apoptotic cells and the mechanisms by which phagocytes recognize and respond to these signals to orchestrate the selective and rapid removal of apoptotic cells. Such unique signals include direct and indirect ‘eat-me’ markers on the apoptotic cell surface, the absence of ‘don't eat-me’ markers normally found on living cells and soluble ‘come-get-me’ signals secreted by apoptotic cells to attract phagocytes to sites of apoptotic cell death. Once apoptotic cells are identified, their uptake by phagocytes further depends on the molecular machinery highly conserved from Caenorhabditis elegans to mammals.