Engulfment Of Apoptotic Cells Research Articles

Engulfment of apoptotic cells involves the redistribution of membrane phosphatidylserine on phagocyte and prey.

Engulfment of apoptotic cells involves the redistribution of membrane phosphatidylserine on phagocyte and prey

Identification of a chromosome 3p14.3-21.1 gene, APPL, encoding an adaptor molecule that interacts with the oncoprotein-serine/threonine kinase AKT2.

AKT2 is a serine/threonine kinase implicated in human ovarian and pancreatic cancers. AKT2 is activated by a variety of growth factors and insulin via phosphatidylinositol 3-kinase (PI3K). However, its normal cellular role is not well understood. To gain insight into the function of AKT2, we performed yeast two-hybrid system to screen for interacting proteins. Using this technique, we identified a novel interactor, designated APPL, which contains a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a leucine zipper, classes of motifs defined in signaling molecules as functional interaction domains with specific targets. The PH domain of APPL shows similarity to those found in GTPase-activating proteins such as oligophrenin-1 and Graf, whereas its PTB domain exhibits homology with CED-6, an adaptor protein that promotes engulfment of apoptotic cells, and IB1, a transactivator of the GLUT2 gene. APPL is highly expressed in skeletal muscle, heart, ovary and pancreas, tissues in which AKT2 mRNA is abundant. APPL interacts with the inactive form of AKT2; moreover, APPL binds to the PI3K catalytic subunit, p110alpha. These data suggest that APPL is an adaptor that may tether inactive AKT2 to p110alpha in the cytoplasm and thereby may expedite recruitment of AKT2 and p110alpha to the cell membrane upon mitogenic stimulation. Furthermore, the APPL gene was mapped to human chromosome 3p14.3-p21.1, where deletions and other rearrangements have often been reported in a variety of tumor types. The identification of APPL may facilitate further analysis of the physiological and oncogenic activities of AKT2.

Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic Neutrophils

Clearance of apoptotic neutrophils (polymorphonuclear leukocyte [PMN]) by macrophages is thought to play a crucial role in resolution of acute inflammation. There is increasing evidence that ingestion of apoptotic cells modulates macrophage behavior. We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself. Rat bone marrow-derived macrophages (BMDM) ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor (TNF) and attenuated by interferon (IFN)-gamma, interleukin (IL)-4, and IL-10. It was inhibitable by the tetrapeptide arg-gly-gln-ser (RGDS), therefore implicating the alphavbeta3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for 30 minutes, 48 hours before rechallenge reduced uptake of apoptotic PMN by 50% compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of preexposure. Comparable and sustained attenuation of uptake was obtained by ligating alphavbeta3 with the monoclonal antibody (MoAb), F11, after a delay of more than 90 minutes, whereas MoAbs to CD25 and CD45 had no effect. Ligation of alpha6beta1 and alpha1beta2, integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11. Therefore, apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.

Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic Neutrophils

AbstractClearance of apoptotic neutrophils (polymorphonuclear leukocyte [PMN]) by macrophages is thought to play a crucial role in resolution of acute inflammation. There is increasing evidence that ingestion of apoptotic cells modulates macrophage behavior. We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself. Rat bone marrow-derived macrophages (BMDM) ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor (TNF) and attenuated by interferon (IFN)-γ, interleukin (IL)-4, and IL-10. It was inhibitable by the tetrapeptide arg-gly-gln-ser (RGDS), therefore implicating the αvβ3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for 30 minutes, 48 hours before rechallenge reduced uptake of apoptotic PMN by 50% compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of preexposure. Comparable and sustained attenuation of uptake was obtained by ligating αvβ3 with the monoclonal antibody (MoAb), F11, after a delay of more than 90 minutes, whereas MoAbs to CD25 and CD45 had no effect. Ligation of α6β1 and α1β2, integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11. Therefore, apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.

CD14 and apoptosis.

In addition to its role as a mediator of innate pro-inflammatory responses following bacterial lipopolysaccharide (LPS) binding, the 55kDa glycosyl-phosphatidylinositol-linked macrophage plasma membrane glycoprotein CD14 is now also known to play a role in phagocytic clearance of apoptotic cells. Although apoptotic cell-associated ligand(s) for CD14 await definition, initial findings suggest that ligand binding occurs close to, or at the same site as, LPS binding. Significantly, in contrast to LPS clearance and in keeping with the non-phlogistic nature of apoptosis, CD14-dependent engulfment of apoptotic cells fails to elicit pro-inflammatory cytokine release from macrophages. Therefore CD14 may be regarded as an innate immune receptor both for microbial products--after binding which activates inflammatory responses--and for self components, which either fail to induce, or alternatively actively suppress, inflammatory responses. Here we review current knowledge of the structure and functions of CD14, its ligands, its possible modes of signal transduction and its place in the panoply of macrophage molecules implicated in apoptotic-cell clearance.

Murine cytomegalovirus induces apoptosis in non-infected cells of the developing mouse brain and blocks apoptosis in primary neuronal culture.

Cytomegalovirus (CMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, we found that apoptosis is induced in the developing mouse brain infected with murine cytomegalovirus (MCMV) in an association with neuronal cell loss. With the combination of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique and immunohistochemical staining, 3.8% of the TUNEL-positive cells were double-stained with the antibody to neuron-specific enolase, while none of the TUNEL-positive cells were stained with antibodies to the immediate early and early viral antigens of MCMV. Furthermore, distribution pattern of the TUNEL-positive cells was different from that of viral DNA-positive cells detected by the in situ DNA-DNA hybridization. More than 30% of the TUNEL-positive cells were double-stained with the F4/80 antibody specific for microglia/macrophages, which were sometimes swollen, presumably the consequence of engulfment of the neuronal apoptotic cells. In the primary neuronal cultures, MCMV infection inhibited the induction of apoptosis either by serum deprivation or by glutamate treatment. It was also confirmed by the double-staining method that apoptosis was not induced in the viral-infected neuronal cultures. These results suggest that MCMV infection induces apoptosis in non-infected neuronal cells, presumably by indirect mechanisms, and that apoptotic cells are engulfed by microglia/macrophages. The induction and blocking of neuronal apoptosis by viral infection may be important for morphological and functional brain disorders in the congenital CMV infection.

Candidate Adaptor Protein CED-6 Promotes the Engulfment of Apoptotic Cells in C. elegans

The rapid engulfment (phagocytosis) of cells undergoing programmed cell death (apoptosis) is a fundamental biological process that is not well understood. Here we report the cloning and functional characterization of ced-6, a gene specifically required for the engulfment of apoptotic cells in the nematode C. elegans. The CED-6 protein contains a phosphotyrosine binding domain at its N terminus and a proline/serine-rich region in its C-terminal half. Genetic mosaic analysis demonstrates that ced-6 acts within engulfing cells. We also show that ced-6 can promote the engulfment of cells at both early and late stages of apoptosis. Our data suggest that CED-6 is an adaptor molecule acting in a signal transduction pathway that specifically mediates the recognition and engulfment of apoptotic cells.

Silica-induced apoptosis in alveolar and granulomatous cells in vivo.

Silica is a toxicant that can stimulate cells to produce various cellular products such as free radicals, cytokines, and growth factors. Silica and its induced substances may induce apoptosis to regulate the evolution of silica-induced inflammation and fibrosis. To examine this hypothesis, groups of Wistar male rats were intratracheally instilled with different doses of Min-U-Sil 5 silica (Silica, Berkeley Springs, WV). Ten days after the instillation, we obtained cells by bronchoalveolar lavage and placed them on slides by cytospin preparation. The slides were stained with Diff-Quik (Lab Aids, Sydney, NSW, Australia) and examined under oil immersion. A substantial number of cells with apoptotic features were identified in all silica-instilled rats and the apoptosis was confirmed by agarose gel electrophoresis. The number of apoptotic cells was clearly related to silica dosage. Engulfment of apoptotic cells by macrophages was also noted. Neutrophil influx in silica-instilled rats could be saturated with the increase of silica dosage and the number of macrophages in different dose groups changed in parallel with the proportion of apoptotic cells. Fifty-six days after instillation, morphologically apoptotic cells could be identified in granulomatous cells of lung tissue from silica-instilled rats. We conclude that intratracheal instillation of silica could induce apoptosis in both alveolar and granulomatous cells, and the apoptotic change and subsequent engulfment by macrophages might play a role in the evolution of silica-induced effects.

A novel flow cytometric method for quantifying phagocytosis of apoptotic cells

Many eukaryotic cell types are capable of specific recognition and phagocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these mechanisms, we designed a novel fluorescence-based method to quantify phagocytosis in vitro using endothelial cell engulfment of apoptotic cells as a model. The B-cell line WEHI-231 was labeled with the fluorophore 5-(&-6)-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA) and then induced to undergo apoptosis by crosslinking cell surface immunoglobulin: An endothelial cell line was subsequently allowed to ingest these TAMRA-labeled apoptotic lymphocytes. After 24 h, non-bound lymphocytes were removed and the monolayers were dissociated. Any nonphagocytosed lymphocytes that remained tightly bound to the endothelial cells were then indirectly immunofluorescein labeled for the pan leukocyte-specific marker CD45. Flow cytometric analysis of the cells distinguished three endothelial cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA+ CD45+); 2) endothelial cells containing phagocytosed apoptotic lymphocytes (TAMRA+ CD45-); and 3) endothelial cells that were not associated with lymphocytes. The identification of these populations was verified by confocal microscopy of sorted cells. The method described herein will facilitate detailed studies on phagocytic recognition of apoptotic cells and should have broad applications to other phagocytic cell systems. Cytometry 27:145–152, 1997. © 1997 Wiley-Liss, Inc.

A novel flow cytometric method for quantifying phagocytosis of apoptotic cells

Many eukaryotic cell types are capable of specific recognition and phagocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these mechanisms, we designed a novel fluorescence-based method to quantify phagocytosis in vitro using endothelial cell engulfment of apoptotic cells as a model. The B-cell line WEHI-231 was labeled with the fluorophore 5-(&-6)-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA) and then induced to undergo apoptosis by crosslinking cell surface immunoglobulin. An endothelial cell line was subsequently allowed to ingest these TAMRA-labeled apoptotic lymphocytes. After 24 h, nonbound lymphocytes were removed and the mono-layers were dissociated. Any nonphagocytosed lymphocytes that remained tightly bound to the endothelial cells were then indirectly immunofluorescein labeled for the pan leukocyte-specific marker CD45. Flow cytometric analysis of the cells distinguished three endothellal cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA+ CD45+); 2) endothelial cells containing phagocytosed apoptotic lymphocytes (TAMRA+ CD45-); and 3) endothelial cells that were not associated with lymphocytes. The identification of these populations was verified by confocal microscopy of sorted cells. The method described herein will facilitate detailed studies on phagocytic recognition of apoptotic cells and should have broad applications to other phagocytic cell systems.

The role of apoptosis in the pathogenesis and treatment of diseases.

In adult multicellular organisms, homeostasis is determined in each cell lineage by a balance between cell death and cell growth. Dysregulation of cell death mechanisms is involved in the pathogenesis of an increasing number of diseases. Defective apoptosis can participate in malignant transformation, viral latency and autoimmune diseases. Excessive apoptotic cell death is involved in CD4+ T-cell depletion observed in acquired immune deficiency syndrome, in fulminant hepatitis associated with infection by hepatitis B and C viruses, in some neurodegenerative disorders and haematological diseases, in polycystic kidney disease and ischaemia. Three steps can be distinguished in the pathway that leads to cell death. The first step involves interactions between the extracellular and intracellular signals that decide whether a cell should live or die. When death is chosen, a common pathway that involves at least the Bcl-2- family of proteins and the interleukin-1 beta (IL-1 beta)-converting enzyme-related cysteine proteases confirms whether or not the cell should die. Finally, if death is allowed to occur, the apoptotic process itself is characterized by deoxyribonucleic acid (DNA) fragmentation, proteolysis and morphological changes that precede the engulfment of apoptotic cells by neighbouring cells and phagocytes. Several inducers and inhibitors of apoptosis acting on one or several of these three steps that characterize the apoptotic process have been identified in vitro. Their potential usefulness in improving the current therapeutic strategies and designing new strategies in several different diseases is discussed.

Human Kupffer Cell Recognition and Phagocytosis of Apoptotic Peripheral Blood Lymphocytes

Cells undergoing apoptosis are recognized and rapidly phagocytosed by macrophages before their degradation, thus preventing the inflammatory reaction and protecting tissues from the damaging effects of released potentially harmful intracellular contents. In spite of growing interest in the mechanisms leading to the engulfment of apoptotic cells, the molecular bases by which an apoptotic cell is recognized are not entirely understood. Among the several potential mechanisms by which a macrophage can identify a cell as apoptotic, the data reported in the present paper support the idea that Kupffer cells phagocytose apoptotic cells by means of lectin-like receptors. Human Kupffer cells, which possess galactose-specific binding sites, can recognize and phagocytose peripheral blood lymphocytes undergoing apoptosis after heat shock (43°C) or cycloheximide treatment, but not normal living peripheral blood lymphocytes. The putative structure by which apoptotic pheripheral blood lymphocytes are targeted as “edible” could be the molecular changes in the plasma membrane. In fact, our experiments indicate that the membranes of apoptotic peripheral blood lymphocytes express increased amounts ofN-acetylgalactosamine,d-galactose, and mannose residues when compared with membranes of normal PBL. Phagocytosis was inhibited by adding to the culture medium sugar cocktail solution (glucose,N-acetylgalactosamine, methyl mannopyranoside, fucose, 80 mMfinal concentration) or to a lower extent by desialylated glycoproteins (lactosylated bovine serum albumin, asialofetuin, 2 mg/ml final concentration), but not by nondesialylated glycoproteins (fetuin, 2 mg/ml final concentration, bovine serum albumin, 20% final concentration). In addition, phagocytosis of apoptotic peripheral blood lymphocytes by human Kupffer cells was a very rapid process, being almost entirely completed within 15 min of incubation.

The time course, localization and quantitation of T- and B-cell mitogen-driven apoptosis in vivo

Apoptosis has very recently been visualized in situ in a mammalian thymus and spleen [1]. We report here the first in situ visualization, localization and quantitation of the time course of mitogen-altered basal levels of apoptosis within the thymus and spleen of a vertebrate. Adult Xenopus leavis, South African clawed toads, were injected intraperitoneally with either the T-cell mitogen, Concanavalin (Con) A, or the B-cell mitogen, lipopolysaccharide (LPS). Controls, reflecting the basal level of apoptosis of both organs, were injected with isotonic phosphate-buffered saline for amphibians (APBS). ConA and LPS failed to enhance the nearly 2% apoptotic rate in the thymic cortex, which is made up largely of immature lymphocytes, but it did double the base level of 2% apoptosis in the mature lymphocytes of the medulla, particularly along the corticomedullary boundary. In the lymphoid splenic white pulp, the 2% basal level was exceeded slightly after ConA treatment, while the 6% basal lymphoid apoptotic rate in the red pulp was enhanced 7-fold in 12 h. LPS induced lymphocytic apoptosis in the partly erythropoietic red pulp of the spleen after 12 h but did not effect the white pulp. Extensive macrophage engulfment of apoptotic cells was apparent in both the thymus and the spleen.