Spontaneous Thymocyte Apoptosis Research Articles

The influence of Ouabain on human dendritic cells maturation.

Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

Hypoxia in the thymus: role of oxygen tension in thymocyte survival.

Our previous studies using oxygen microelectrodes showed that the thymus is grossly hypoxic under normal physiological conditions. We now have investigated how oxygen tension affects the thymus at the cellular and molecular level. Adducts of the hypoxia marker drug pimonidazole accumulated in foci within the cortex and medulla and at the corticomedullary junction, consistent with the presence of widespread cellular hypoxia in the normal thymus. Hypoxia-associated pimonidazole accumulation was decreased but not abrogated by oxygen administration. Genes previously reported to be induced by hypoxia were expressed at baseline levels in the normal thymus, indicating that physiological adaptation to hypoxia occurred. Despite changes in thymus size and cellularity, thymic PO(2) did not change with age. Combined assays for hypoxia and cell death showed that hypoxia achieved using either hypoxic gas mixtures or high-density culture in normoxia decreased spontaneous thymocyte apoptosis in vitro. Taken together, these data suggest that regulatory mechanisms exist to maintain thymic cellular hypoxia in vivo and that oxygen tension may regulate thymocyte survival both in vitro and in vivo.

Effect of 17β-estradiol and voluntary exercise on lymphocyte apoptosis in mice

During the perimenopause, women may begin estrogen replacement therapy (ERT) and physical activity programs to reduce the symptoms of the climacteric. High-intensity exercise increases lymphocyte apoptosis, and estrogen is also known to have immune modulatory effects. The present study determined whether (1) estrogen exposure in vivo, and (2) low-intensity, voluntary exercise affect thymic and splenic lymphocyte apoptosis in adult female mice. ‘Middle-aged’ (>1 year), ovarian-intact female B6D2F 1 mice were implanted with 17β-estradiol (E) pellets (L: 3 μg/day or H: 12 μg/day) or placebo (P: 0 μg/day). Mice were given 1 week to recover from implantation surgery after which they were randomized to wheel-running or no-wheel-running conditions. Twenty one days later, mice were sacrificed and thymus and spleen removed for determination of percent apoptosis and percent necrosis by flow cytometry, serum E levels by RIA, and tissue and body weights. Estrogen-treated, ovarian-intact mice accumulated less cumulative wheel-running activity than mice implanted with placebo ( P<.001). E exposure was associated with lighter thymuses ( P<.05), higher thymocyte apoptosis ( P<.001), and higher serum E levels ( P<.001), effects which were not modified by voluntary exercise. In contrast, splenocyte apoptosis and spleen weights did not differ by estrogen treatment or exercise. The results suggest that in vivo exposure to supplemental estrogen is associated with greater spontaneous apoptosis of thymocytes and reduced thymus weights in older ovarian-intact mice. The clinical significance for thymic (cellular) immunity in perimenopausal women given HRT remains to be determined.

Spontaneous thymocyte apoptosis is regulated by a mitochondrion-mediated signaling pathway.

Most thymocytes that have not successfully rearranged their TCR genes or that express a receptor with subthreshold avidity for self-Ag/MHC enter a default apoptosis pathway, death by neglect. Spontaneous thymocyte apoptosis (STA), at least in part, may mimic this process in vitro. However, the molecular mechanism(s) by which thymocytes undergo this spontaneous apoptosis remains unknown. Here, we report that caspsase-1 and caspase-3 are activated during STA, but these caspases are dispensable for this apoptotic process. The inhibition of STA by a pan-caspase inhibitor, zVAD, suggests that multiple caspase pathways exist. Importantly, the early release of cytochrome c from mitochondria closely correlates with the degradation of Bcl-2 and Bcl-xL and a decrease in the ratios of Bcl-2 and Bcl-xL to Bax during STA. These findings suggest that the degradation of Bcl-2 and Bcl-xL may favor Bax to induce cytochrome c release from mitochondria, which subsequently activates downstream caspases in STA. Our data provide the first biochemical insight into the molecular mechanism of STA.

Nuclear RNA is extruded from apoptotic cells.

During spontaneous apoptosis of thymocytes there is extrusion of ribonucleoproteins (RNPs) from the cell. The aim of this investigation was to elucidate whether the RNP aggregates in apoptotic cells and bodies still contain RNA in an appreciable amount. We demonstrated by specific cytochemical techniques that the aggregates of nuclear RNPs extruded in the cytoplasm of spontaneously apoptotic thymocytes contain RNA in a sufficient amount to be detected cytochemically. These heterogeneous ectopic RNP-derived structures (HERDS) are formed by perichromatin fibrils, interchromatin granules, perichromatin granules, and nucleolar material. The RNA detected inside these clusters should therefore correspond to both mRNA and snRNA as well as to rRNA. We never observed DNA-containing aggregates in the cytoplasm of apoptotic thymocytes. The presence of RNA in the HERDS that may be released from apoptotic cells suggests that the decrease in the amount of total RNA during apoptosis may be mostly linked to cellular extrusion rather than to degradation of RNA by RNase activities. Another interesting aspect of these results lies in the hypothesis of apoptosis as a possible cause for the presence of autoantibodies in the serum of patients with systemic autoimmune diseases.

Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

Still Immunodetectable Nuclear RNPs Are Extruded from the Cytoplasm of Spontaneously Apoptotic Thymocytes

The fate of different nuclear ribonucleoprotein (RNP) components was investigated during spontaneous apoptosis of thymocytes using specific monoclonal antibodies against snRNPs, hnRNPs, and ribosomal proteins at light and electron microscopy levels and by flow cytometry. It was found that, during apoptosis, nuclear RNP-containing structures (perichromatin granules, interchromatin granules, and perichromatin fibrils) segregate in the interchromatin space and cluster into heterogeneous aggregates of granules in which some of the structures may still be recognized morphologically. Along with the progress of apoptosis, the clusters are extruded from the nucleus into the cytoplasm, from which they are finally released via cytoplasmic extrusions. At all these stages, RNPs inside the clusters are always recognized by specific antibodies, even when they bleb out of the cell surface, thus suggesting that degradation of RNPs might be only partial during apoptosis. This could be potentially harmful in genetically susceptible subjects, as the appropriate MHC class II molecules may capture and present normally cryptic self-peptides. It is tempting to speculate that this event might have implications in the etiology of autoimmune diseases.

Nuclear ribonucleoprotein-containing structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A morphological study by electron microscopy.

The rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during spontaneous thymocyte apoptosis has been investigated by electron microscopy. Along with chromatin margination and condensation. RNPs segregate into the central portion of the nucleus, in the form of heterogeneous clusters of granules which contain interchromatin and perichromatin granules, perichromatin fibrils, nucleolar components, and probably coiled bodies. In parallel with progressive chromatin condensation and karyorrhexis, granule clusters are then extruded into the cytoplasm and are finally released at the cell surface as membrane-bound cytoplasmic debris, sometimes in association with apparently undamaged organelles such as centrioles. It is likely that this RNP segregation may correlate with a severe impairment of protein synthesis. A similar phenomenon was observed in elongating spermatids, when transcriptional arrest is induced during the process of reversible silencing of the male genome. It may be hypothesized that segregation into heterogeneous granule clusters could be a common mechanism to remove redundant RNP-containing structures from the cell.

Spontaneous Apoptosis of Thymocytes Is Uncoupled with Progression through the Cell Cycle

Most developing lymphocytes spontaneously die in the thymus during positive and negative selection of the T cell repertoire. By evaluating the expression of the proliferation antigens Ki-67 and PCNA, we demonstrated here that more than 95% of thymocytes are potentially proliferating. The coincidence within the same cell population of death and proliferation is thus apparent in developing thymocytes. Using dual-parameter cytometric techniques to evaluate in single cells the amount of DNA versus light-scattering values, we found that spontaneous thymocyte apoptosis occurs with similar frequency in all the cycle phases, whereas apoptosis induced by the anti-topoisomerase-II, etoposide (which is the consequence of irreversible DNA damage), takes place with higher frequency in S and G2phases (i.e., in those cycle phases in which DNA is subjected to torsional constraints). The capability of thymocytes to enter apoptosis was also monitored by digesting DNAin situwith DNase I (a nuclease that cleaves DNA mimicking the nuclear damage common to most apoptotic suicides). We also show that endonuclease-mediated DNA digestion occurs to a similar extent in cells with different DNA contents, i.e., in cycle phases in which the superstructural organization of chromatin is markedly different.

Activation of the Na(+)/H(+) antiporter, Na+/HCO3(-)/CO3(2-) cotransporter, or Cl(-)/HCO3(-) exchanger in spontaneous thymocyte apoptosis.

This study was undertaken to define the role of ion transporters on the apoptosis of thymocytes. Culture conditions, such as the ionic strength of NaCl, Ca2+, the buffer system (HCO3-/CO2), and the pH, could influence the spontaneous apoptosis of thymocytes. Depletion of NaCl in the culture medium either delayed or completely inhibited the apoptotic process of thymocytes, while its restoration led to a dose-dependent apoptosis. A high concentration (100 microM) of Ca2+ induced thymocyte apoptosis in the nominal absence of NaCl, whereas a low concentration (10 microM) enhanced apoptosis in the presence of 138 mM NaCl. Thymocytes had a higher spontaneous apoptotic rate in cultures without HCO3-/CO2 than in those with HCO3-/CO2. The thymocyte apoptosis completely ceased in medium at pH 6.0 and was considerably enhanced at pH 7.6. Intracellular pH, determined with the pH-sensitive bis-carboxyethyl carboxyfluorescein probe, was higher in apoptotic thymocytes than in nonapoptotic cells. Spontaneous apoptosis occurred in cells with alkaline intracellular condition, whereas it was considerably retarded in cells under acidified conditions. Amiloride analogue, including 5-(N,N-dimethyl)-amiloride (Na+/H+ antiporter), 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (Cl-/HCO3- exchanger), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (Na+/HCO3-/CO3(2-) cotransporter), inhibited the spontaneous thymocyte apoptosis. In contrast, neither cycloheximide nor actinomycin D had the same effect. In addition, thymocyte apoptosis was enhanced by PMA, but inhibited by forskolin. Taken together, thymocytes cultured in vitro underwent apoptosis with increased intracellular pH via activation of Na+/H+ antiporter, Na+/HCO3-/CO3(2-) cotransporter, or Cl-/HCO3- exchanger. This process does not require de novo protein synthesis.

Characterization of the spontaneous apoptosis of rat thymocytes in vitro.

Unlike splenic or blood lymphocytes, rat thymocytes spontaneously undergo continuously increasing apoptosis during culture. In this study we characterized apoptotic thymus cells of rats according to cell size, nuclear dye binding and surface marker expression. Furthermore, the effects of cell density in culture, the age of the donor animals, glucocorticoids, and inhibition of protein synthesis were studied. It was found that: (1) apoptotic rat thymocytes are recognized in flow cytometry as small, acridine orange low, CD4low, CD8high cells; (2) the rate of apoptosis is dependent on the cell density in the culture in a biphasic manner; (3) thymic apoptosis increases with age of the donor animal in fresh, as well as in 24-hour cultivated cell suspensions; (4) neither adrenalectomy nor in vivo or in vitro treatment with the glucocorticoid antagonist RU 38486 influenced spontaneous apoptosis of thymocytes, and (5) inhibition of protein synthesis, which decreases apoptosis induced by corticosterone, had no effect on spontaneous apoptosis of thymocytes.

Cadmium in the Environment

Most thymocytes that have not successfully rearranged their TCR genes or that express a receptor with subthreshold avidity for self-Ag/MHC enter a default apoptosis pathway, death by neglect. Spontaneous thymocyte apoptosis (STA), at least in part, may mimic this process in vitro. However, the molecular mechanism(s) by which thymocytes undergo this spontaneous apoptosis remains unknown. Here, we report that caspsase-1 and caspase-3 are activated during STA, but these caspases are dispensable for this apoptotic process. The inhibition of STA by a pan-caspase inhibitor, zVAD, suggests that multiple caspase pathways exist. Importantly, the early release of cytochrome <i>c</i> from mitochondria closely correlates with the degradation of Bcl-2 and Bcl-x_L and a decrease in the ratios of Bcl-2 and Bcl-x_L to Bax during STA. These findings suggest that the degradation of Bcl-2 and Bcl-x_L may favor Bax to induce cytochrome <i>c</i> release from mitochondria, which subsequently activates downstream caspases in STA. Our data provide the first biochemical insight into the molecular mechanism of STA.