Apoptotic Thymocytes Research Articles

Characterization of apoptosis-like endonuclease activity in avian thymocytes

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricar☐ylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.

Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. NUC18 is not histone H2B.

Glucocorticoids stimulate apoptosis in rat thymocytes that is characterized by internucleosomal DNA degradation. We have previously identified an 18-kDa calcium-dependent nuclease whose activity is associated with this DNA degradation. The existence of this nuclease has been challenged by Alnemri and Litwack (1989) J. Biol. Chem. 264, 4104-4111, who suggest that the nuclease we observed was histone H2B. We report here a modified nuclease assay which uses [32P] DNA as a substrate that has enabled the purification and characterization of the 18-kDa nuclease (NUC18). Using Bio-Rex 70 chromatography in conjunction with this assay, we show that NUC18 can be separated from histone H2B. Enzymatically active NUC18, purified to apparent homogeneity, failed to react with two different anti-histone H2B antibodies. NUC18 was inactive in the absence of calcium and known inhibitors of apoptosis, i.e. zinc and aurintricarboxylic acid inhibit its activity. Although NUC18 activity was detected in nuclear extracts of thymocytes of both control and glucocorticoid-treated thymocytes, these activities were distinct. Gel filtration analysis revealed that NUC18 was present as a high molecular weight complex (greater than 100 kDa) in both groups of cells, whereas it also existed as a low molecular weight form in glucocorticoid-treated cells. Thus, NUC18 remains a candidate for the endonuclease responsible for the DNA degradation component of the apoptotic process.

Internucleosomal Deoxyribonucleic Acid Cleavage Activity in Apoptotic Thymocytes: Detection and Endocrine Regulation*

Apoptosis is a programmed form of cell death that occurs under numerous physiological conditions, including endocrine regulation of specific cell populations. We have investigated the biochemical mechanisms involved in glucocorticoid-induced apoptosis in rat thymocytes. Internucleosomal cleavage of chromatin into oligonucleosomal fragments is common to all forms of apoptosis and precedes the onset of cell death. To identify the endonuclease that is responsible for the specific pattern of DNA degradation in glucocorticoid-induced apoptosis, we have developed an assay to measure internucleosomal cleavage activity in thymocyte nuclear extracts. This assay uses nuclei from cells resistant to hormone-induced DNA fragmentation (HeLa cells) as a substrate for nuclear extracts prepared from thymocytes of adrenalectomized rats treated with either dexamethasone (dex) or vehicle (control). After incubation at room temperature for 90 min, the HeLa DNA is purified, and its integrity is analyzed by agarose gel electrophoresis. The appearance of internucleosomal fragments of HeLa DNA is indicative of nuclease activity in the thymocyte nuclear extract. Nuclear extracts prepared from thymocytes of rats treated with dex for 5 h caused internucleosomal cleavage of HeLa DNA, whereas extracts from control rats did not result in any DNA fragmentation. Regulation of nuclease activity by dex was time dependent. Internucleosomal cleavage activity in thymocyte extract from dex-treated animals was detected as early as 2 h after hormone treatment and occurred before any detectable change in cell viability. Maximal extractable nuclease activity was coincident with decreased thymocyte viability and thymic involution. In contrast, extracts from medullary thymocytes, which are the only thymocytes that survive 72 h of glucocorticoid treatment, did not contain nuclease activity by this assay. Regulation of internucleosomal cleavage activity by dex was dose dependent and was specific for the glucocorticoid class of steroid hormones. Furthermore, the dex-induced response was inhibited by pretreating rats with the glucocorticoid receptor antagonist RU486, indicating that receptor-mediated processes are involved in the regulation of nuclease activity. The similarities between the regulation of internucleosomal cleavage activity reported here and the previously described degradation of thymocyte DNA in vivo makes this nuclease a likely constituent of the apoptotic process.

Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes: NUC18 is not histone H2B

Identification, purification, and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes: NUC18 is not histone H2B

Phagocytosis of aged human neutrophils by macrophages is mediated by a novel "charge-sensitive" recognition mechanism.

The removal of neutrophils and their histotoxic contents from the inflamed site is a prerequisite for resolution of tissue injury, and a point at which factors critical to the pathogenesis of chronic inflammation may act. Engulfment of intact, senescent neutrophils by macrophages represents an important neutrophil disposal process. In this study the mechanism by which human monocyte-derived macrophages (M phi) recognized and ingested human neutrophils that had been aged in culture was studied using an in vitro phagocytic assay. Inhibition of M phi receptors for Ig Fc and the opsonic complement fragments C3b and iC3b with MAbs to M phi FcR, CR1, CR3, and CR4 had no effect on recognition, and the pattern of inhibition observed when polyanions were included in the medium at 1 mg/ml was different from that reported for the M phi receptor for protein advanced glycosylation end products (AGE), indicating a recognition mechanism different from those proposed for M phi phagocytosis of senescent erythrocytes. Furthermore, although aging neutrophils undergo programmed cell death (or apoptosis), which is directly related to recognition by M phi, the pattern of inhibition observed with monosaccharides was different from that reported to inhibit the binding of apoptotic mouse thymocytes to isologous M phi. By contrast, evidence was obtained for a novel recognition mechanism inhibitable by cationic sugars and amino acids in a charge-dependent fashion, and directly modulated by pH but not affected by inhibitors of the mannose-6-phosphate, sheep erythrocyte, mannosyl-fucosyl, asialoglycoprotein, and scavenger receptors of the macrophage. These observations suggest that hydrogen ions and charged molecules may modulate M phi uptake of senescent neutrophils at inflamed sites, and that recognition itself may involve charged structures on the cells.

Properties of embryo-derived platelet activating factor

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.

Naloxone inhibits development of preimplanatation mouse embryos

There is evidence that apoptotic cells and oxidized low density lipoprotein (oxLDL) particles have common ligands on their surface consisting of oxidized phospholipids which bind to scavenger receptors in macrophages leading to phagocytosis. Some effects of oxLDL binding to its receptor(s) were shown to be inhibited by Platelet Activating Factor (PAF)-receptor antagonists. Thus, we investigated the effect of PAF-receptor antagonists on the phagocytosis of apoptotic, necrotic and viable thymocytes by murine peritoneal macrophages. It was found that phagocytosis of altered cells is significantly increased compared to viable cells, a phenomenon reversed by pre-treatment of macrophages with PAF-receptor antagonists (WEB2170 and CV3988), PAF or oxLDL. Phagocytosis of altered cells induced negligible expression of cyclooxygenase-2 (COX-2) but strongly potentiated the LPS-induced expression of this enzyme. This phenomenon was restricted to altered cells and was reversed by pre-treatment of macrophages with PAF-receptor antagonists. These findings indicate that apoptotic and necrotic cells share common ligands with PAF and oxLDL and suggest the involvement of PAF-like receptors in the enhanced clearance of these cells.