Rat Thymocytes Research Articles

Analysis of 15-Lipoxygenase Activity in Irradiated Thymocytes

We studied the effect of gamma-radiation on 15-lipoxygenase activity in rat thymocytes. The enzyme activity was determined as the rate of linoleic acid oxidation by the protein fraction isolated from the control and irradiated thymocytes under standard conditions. We demonstrate lipoxygenase activation immediately after irradiation of thymocytes. High lipoxygenase activity is observed in the cells for no more than an hour after irradiation. No high lipoxygenase activity later indicates that its synthesis is not directly induced by irradiation. Irradiation-induced generation of lipid peroxides can be the factor of lipoxygenase activation.

The structure?activity relationship of various YO compounds, novel plasmin inhibitors, in the apoptosis induction

We have previously reported that YO-2, a selective plasmin inhibitor, induces thymocyte apoptosis. To elucidate the mechanism of YO-2-induced apoptosis, other YO compounds with different plasmin inhibitory action were tested for the pro-apoptotic activity in this study. The treatment of rat thymocytes with the YO compounds which had the hydrophobic but not the hydrophilic moiety at the C-terminal increased DNA fragmentation, the number of condensed nuclei and caspase-3-like activity. All pro-apoptotic YO compounds not only were potent plasmin inhibitors but also had the hydrophobic C-terminal as the common structure. Therefore, the target molecule of the YO compounds may be located not on the cell surface but rather inside the cells.

Effect of ghrelin on the apoptotic deletion rate of different types of cells cultured in vitro

Evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, controls the growth of several human and rat cell types cultured in vitro. Hence, we have investigated, by using both TUNEL and ELISA assays, the effects of 10(-8) M ghrelin on the basal apoptotic deletion rate of rat osteoblasts and thymocytes, rat and human adrenocortical cells, human umbilical vein endothelial cells, and human aldosteronoma cells cultured in vitro, as well as of the human adrenocortical carcinoma-derived cell lines NCI-H295 and SW-13. Both assays consistently showed that ghrelin did not affect apoptotic rate of normal rat and human cells, but significantly enhanced apoptotic deletion in aldosteronoma, NCI-H295 and SW-13 cell cultures. Due to the central role of apoptosis in the control of tumor growth, these findings, if confirmed in other tumor cell types, could suggest an antitumoral action of ghrelin.

Terfenadine induces thymocyte apoptosis via mitochondrial pathway

The treatment of rat thymocytes with 10 μM terfenadine resulted in a significant increase in DNA fragmentation. The DNA fragmentation induced by terfenadine was dependent on its concentration and incubation time. In terfenadine-treated cells, the translocation of phosphatidylserine from the inside of plasma membrane to the outside, an early event of the apoptotic process, and chromatin condensation, the morphological characterization of apoptotic cell death, were observed. Terfenadine stimulated caspase-8, -9 and -3-like activities in an incubation time-dependent manner in thymocytes. The active forms of caspase-3 and -9 were detected in the extract from terfenadine-treated cells by immunoblotting analysis using specific antibodies to caspases, but active caspase-8 was not found in this fraction. Decrease in mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol were observed in terfenadine-treated thymocytes. These results suggest that terfenadine induces apoptosis in rat thymocytes via mitochondrial pathway.

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes

Inhibition of Notch signaling biases rat thymocyte development towards the NK cell lineage.

Notch receptors are involved in directing the choice between alternative cell fates in developmental scenarios such as thymopoiesis. By pharmacological interference in rat fetal thymus organ culture we show that inhibition of Notch signaling arrests T cell development at an early double-negative stage and is accompanied by a dramatic increase in the number of NK cells. These cells show an activated phenotype, lack recombination of the TCR beta gene locus and express perforin. Similarly, in thymic lobes reconstituted with fetal liver cells, progenitors predominantly develop into NK cells both after pharmacological interference of Notch and after treatment with a recombinant rat Notch1/Fc chimera. Collectively, this identifies the lineage decision of NK/T precursor cells as an important site of Notch action in rat thymocytes.

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes.

The effects of polysorbate 80, a non-ionic surfactant widely used in pharmaceutical products, on rat thymocytes were examined to reveal its toxic property at the cellular level. Polysorbate 80 at concentrations of 1-100 microg/ml did not significantly affect the cell viability. This surfactant at 30 microg/ml or more augmented the intensity of fluo-3 fluorescence, indicating the increase in intracellular Ca(2+) concentration. Such an augmentation of fluo-3 fluorescence by polysorbate 80 was not seen under the Ca(2+)-free condition, suggesting that polysorbate 80 increased membrane Ca(2+) permeability. The concentration-dependent polysorbate 80 at 10 microg/ml or more attenuated the intensity of 5-chloromethylfluorescein, indicating a decrease in cellular content of glutathione by polysorbate 80. Furthermore, the agent at 1 microg/ml or more attenuated the intensity of bis-(1,3-dibutylbarbituric acid) trimethine oxonol fluorescence, being independent from the changes in membrane potential. This phenomenon indicates that polysorbate 80 at 1 microg/ml or more may attenuate the incorporation of anionic compounds into the membranes. It can be suggested that polysorbate 80 modifies some of membranes and intracellular physiological parameters without affecting the cell viability.

Neonatal Capsaicin Treatment Affects Rat Thymocyte Proliferation and Cell Death by Modulating Substance P and Neurokinin-1 Receptor Expression

Herein we provide evidence that substance P (SP) and its neurokinin-1 receptor (NK-1R) expressed on thymocytes counteract thymus depletion induced by neonatal capsaicin (CPS) treatment by affecting thymocyte proliferation and apoptotic death. SP administration reversed the CPS-mediated inhibitory effects on the total thymocyte number and subset distribution, namely CD4+ and CD4– CD8– cells, through its interaction with NK-1R as shown by concomitant NK-1R (SR140333) antagonist administration. SP-induced enhancement of thymus cellularity parallels its ability of inhibiting the thymocyte apoptotic program. Indeed, exogenously administered SP completely nullified CPS-induced apoptosis, and SR140333 abrogated the SP-mediated protective effect. SP administration also stimulated concanavalin A (Con A)-induced thymocyte proliferation of CPS-treated rats, completely reversing the CPS-induced inhibition. The SP-mediated stimulation of Con A-induced thymocyte proliferation was NK-1R dependent as shown by concomitant administration of SP and SR140333 to CPS-treated rats. Our results also demonstrate that CPS treatment induces a marked decrease of thymocyte PPT-A mRNA level and endogenous SP content as evaluated by quantitative RT-PCR, in situ hybridization and cytofluorimetric analysis. By contrast, NK-1R mRNA levels were increased in thymocytes from CPS-treated rats. Exogenous SP administration augmented PPT-A, SP and NK-1R thymocyte expression in CPS-treated rats, and this enhancement was antagonized by SR140333 administration. Overall, our results strongly suggest that the immunomodulatory effects of neonatal CPS treatment on rat thymocyte functions are dependent on vanilloid-mediated regulation of SP and NK-1R functional expression by neuronal and immune cells.

Property of thimerosal-induced decrease in cellular content of glutathione in rat thymocytes: a flow cytometric study with 5-chloromethylfluorescein diacetate

There is a concern on the part of public health community that adverse health consequences by thimerosal, a preservative in vaccines for infants, may occur among infants during immunization schedule. Therefore, the effect of thimerosal on cellular content of glutathione was examined on thymocytes obtained from 4-week-old rats using a flow cytometer and 5-chloromethylfluorescein diacetate. Thimerosal at concentrations ranging from 1 to 10 μM reduced the cellular content of glutathione in a concentration-dependent manner, and the complete depletion of cellular glutathione was observed when the cells were treated with 30 μM thimerosal. L-Cysteine significantly attenuated the actions of thimerosal to reduce the glutathione content and to increase the intracellular Ca 2+ concentration. Prolonged incubation (24 h) with 1–3 μM thimerosal induced the apoptosis. The cytotoxic action of thimerosal was greatly augmented when the cells suffered oxidative stress induced by H 2O 2. It may be unlikely that thimerosal exerts potent cytotoxic action under the in vivo condition because the blood concentration of thimerosal after receiving vaccines does not seem to reach micromolar range and nonprotein thiols at micromolar concentrations are present in the blood.

Fluorometric evidence for different stoichiometries for the Na+/Mg2+ exchange in Mg-loaded rat thymocytes

The regulation of the cytosolic free magnesium concentration ([Mg2+]i) is a fundamental cellular process that requires magnesium extruding mechanisms. Here, we present evidence indicating that rat thymocytes are endowed with different Na/Mg exchange systems. Fluxes of magnesium were measured using the fluorescent magnesium indicator magfura-2. Cells were loaded with magnesium using the calcium ionophore A-23187 to 0.6-8.0 mM [Mg2+]i (resting [Mg2+]i = 0.38 +/- 0.06 mM, n = 5). The presence of extracellular sodium was required for magnesium exit. The initial rate of [Mg2+]i was stimulated by extracellular sodium with Michaelis-Menten kinetics. The Vmax of the sodium-dependent magnesium exit was markedly increased by [Mg2+]i. Holding the membrane potential at either -84 mV or at -10 mV had different effects on the sodium-stimulated magnesium efflux, depending on the extracellular sodium concentration ([Na+]o). At 10-30 mM [Na+]o, the magnesium efflux was faster at -10 mV than at -84 mV. Conversely, at 50-200 mM [Na+]o, the efflux of magnesium was faster at -84 mV that at -10 mV. At 75 mM [Na+]o, the activities where nearly the same at both membrane potential values. These observations suggest that the stochiometry of the Na+/Mg2+ exchange changes with [Na+]o.

The Effect on Rat Thymocytes of the Simultaneous In Vivo Exposure to 50-Hz Electric and Magnetic Field and to Continuous Light

Thymus plays an important role in the immune system and can be modulated by numerous environmental factors, including electromagnetic fields (EMF). The present study has been undertaken with the aim to investigate the role of long-term exposure to extremely low frequency electric and magnetic fields (ELF-EMF) on thymocytes of rats housed in a regular dark/light cycle or under continuous light. Male Sprague-Dawley rats, 2 months old, were exposed or sham exposed for 8 months to 50-Hz sinusoidal EMF at two levels of field strength (1 kV/m, 5 μT and 5 kV/m, 100 μT, respectively). Thymus from adult animals exhibits signs of gradual atrophy mainly due to collagen deposition and fat substitution. This physiological involution may be accelerated by continuous light exposure that induces a massive death of thymocytes. The concurrent exposure to continuous light and to ELF-EMF did not change significantly the rate of mitoses compared to sham-exposed rats, whereas the amount of cell death was significantly increased, also in comparison with animals exposed to EMF in a 12-h dark-light cycle. In conclusion, long-term exposure to ELF-EMF, in animals housed under continuous light, may reinforce the alterations due to a photic stress, suggesting that, in vivo, stress and ELF-EMF exposure can act in synergy determining a more rapid involution of the thymus and might be responsible for an increased susceptibility to the potentially hazardous effects of ELF-EMF.

Ortho-Substituted PCBs Kill Thymocytes

The effects of exposure of acutely dissociated rat thymocytes to various polychlorinated biphenyl (PCB) congeners were examined using flow cytometry. Non-planar, ortho-substituted congeners caused a rapid cell death at low micromolar concentrations, while coplanar, dioxin-like congeners at the same concentration were without significant effect. The most potent of the congeners studied was PCB 52 (2,2',5,5'-tetrachlorobiphenyl), which had an IC50 of 3.96 microM at 20 min. Prior to loss of viability there was a decrease in mitochondrial membrane potential Delta Psi m, an accumulation of intracellular calcium, and a progressive leakiness of the plasma membrane. Application of PCB 52 in calcium-free medium reduced the calcium accumulation, but did not reduce cell death. Agents that depolarized mitochondria also did not induce the same degree of cell death caused by PCB 52. Cyclosporin A, which prevents opening of the mitochondria permeability transition channel, protected against cell death but did not protect against mitochondrial depolarization or calcium accumulation. Rapamycin and FK 506 at high concentration provided partial protection against cell death. These observations indicate that the ortho-substituted PCB 52 disrupts plasma, mitochondrial and endoplasmic reticulum membranes. We hypothesize that PCB 52 incorporates into lipid bilayers and with its bulky, three-dimensional ortho-substituted congener structure disrupts membrane function to a greater degree than coplanar congeners.

Increase in number of annexin V-positive living cells of rat thymocytes by intracellular Pb 2+

Lead is ubiquitous in our environment and lead poisoning is a major public health problem worldwide. In this study, to see if intracellular Pb 2+ induces the exposure of phosphatidylserine in rat thymocyte membranes, we have examined the effect of PbCl 2 on rat thymocytes treated with A23187 using a flow cytometer with appropriate fluorescent indicators under nominally-Ca 2+-free condition. PbCl 2 at 1–30 μM dose-dependently induced the exposure of phosphatidylserine on outer membranes, associated with increasing the concentration of intracellular Pb 2+. The potency of intracellular Pb 2+ to induce the apoptotic change in thymocyte membranes seems to be greater than those of intracellular Ca 2+ and Cd 2+. Results suggest that intracellular Pb 2+ triggers apoptosis of rat thymocytes. This action of Pb 2+ may be one of mechanisms for the lead-induced changes in immunity.

Tri- n-butyltin-induced cytotoxicity on rat thymocytes in presence and absence of serum

Influence of fetal bovine serum (FBS) on cytotoxicity induced by tri- n-butyltin (TBT), an environmental pollutant, on rat thymocytes was examined to reveal how FBS modifies TBT cytotoxicity. As the medium FBS concentration was increased from 0 to 10%, the cytotoxicity of TBT was dose-dependently reduced when the cells were incubated with 1 μ m TBT for 3 h. Almost complete inhibitions of TBT-induced changes in cell viability and population of cells with exposed phosphatidylserine (cells undergoing apoptosis) were observed when the FBS concentration was 10%. Thus, the cytotoxicity induced by 3 h incubation with TBT in FBS-free medium may be different from that in medium containing 10% FBS. However, even in presence of 10% FBS, TBT at concentrations ranging from 10 to 300 n m exerted cytotoxic action on rat thymocytes when the cells were incubated with TBT for 24 h. TBT dose-dependently increased the population of shrunken cells, of which more than 30% were stained with propidium. TBT at 30 n m or more significantly increased the population of cells with hypodiploid DNA, indicating TBT-induced apoptotic cell death. Thus, in the presence of 10% FBS, the prolonged incubation (24 h) of rat thymocytes with TBT at nanomolar concentrations induced apoptosis rather than necrosis.

Characterization of immunomodulatory polysaccharides from Salvia officinalis L.

Crude polysaccharide fractions, rich mainly in arabinogalactans (A), pectin (B) and glucuronoxylan-related polymers (D), have been obtained from aerial parts of sage ( Salvia officinalis L.) by sequential extraction with various reagents. Arabinogalactans displayed on HPLC a dominance of lower molecular-mass polymers ( M ̄ w <10 ,000), while pectin and glucuronoxylan-related polysaccharides showed predominance of polymers with M ̄ w >50 ,000. Individual polysaccharide fractions were examined for their immunomodulatory activity in the in vitro comitogenic thymocyte test. The polysaccharide fractions tested possessed the capacity to induce rat thymocyte proliferation in the order D>B>A. Besides, fraction D possessed a significant comitogenic effect, and the SI comit/SI mit ratio 3–4 indicates potential adjuvant properties of this glucuronoxylan-rich material.

DNA supercoiling in apoptotic chromatin

In a previous paper, we have reported that in rat thymocyte apoptosis chromatin undergoes a specific structural change as well as an appreciable increase in the unacetylated forms of histones H3 and H4. Here, we show that H3 and H4 deacetylation bears no relation to chromatin condensation, and present new ultrastructural and topological observations that largely clarify the organization of the condensed state. The texture of the latter corresponds to a closely woven network of negatively supercoiled 11 nm fibers, as shown by both ultrastructural observations and relaxation experiments using ethidium bromide. Circularly closed chromatin loops undergoing apoptotic condensation, clearly showing nucleosome compact dimers or higher oligomers, as well as long stretches of supercoiled DNA, have also been detected. All of these modifications are strongly reminiscent of the alterations induced in nucleosome bearing plasmids by the chromatin remodeling factors SWI/SNF and RSC.

Massive telomere loss and telomerase RNA expression in dexamethasone-induced apoptosis in mouse thymocytes

Apoptosis is a well-recognized process of cell death occurring under several physiological and pathological conditions and represents the principal mechanism involved in cell selection in the thymus. Glucocorticoids are well known to stimulate apoptosis in rat thymocytes. However, it is unclear whether the same changes occur after in vivo glucocorticoid treatment in mice. Chromosomal stability and cell viability require a proficient telomeric end-capping function. Cells with critical telomere shortening and telomerase dysfunction undergo increased apoptosis. In turn, the change in telomere function in cells undergoing apoptosis is not fully characterized. In order to investigate this, we studied the changes in thymocytes after dexamethasone administration in BALB/c mice. The loss of normal thymocytes coincided with the appearance of small dense cells with characteristic features of apoptosis including condensed chromatin, internucleosomal DNA cleavage, and a “hypodiploid” peak on flow cytometry, which suggested that dexamethsone-induced thymocyte apoptosis in BALB/c mice could be considered a well-defined experimental model for studying apoptotic processes. Dexamethasone-treated thymocytes exhibited rapid and dynamic loss of telomeric sequences and up-regulation of telomerase RNA as an early event in the apoptotic process. Telomerase activity was unchanged in this event. Thereafter, telomere gain associated with an increase in telomerase activity occurred in the regenerative process of the thymus. These results suggest a role of telomere loss and up-regulation of telomerase RNA as key apoptosis sensors.

Optimization of enhancement of therapeutic efficacy of ultrasound: Frequency-dependent effects on iodine formation from KI-starch solutions and ultrasound-induced killing of rat thymocytes.

We investigated liberation of iodine from solutions of KI-starch and cell lysis of rat thymocytes in argon-and nitrous oxide-saturated aqueous solutions induced by ultrasound at frequencies of 38 and 500 kHz and 1 and 2 MHz. Iodine was liberated in argon-saturated solutions exposed to ultrasound at 38 kHz, 500 kHz, and 1 MHz but not at 2 MHz. Lysis occurred in argon-saturated solutions at all four frequencies, but only at 38 kHz in nitrous oxide-saturated cell suspensions. No iodine was liberated in the other nitrous oxide-saturated samples. Relative ratio of the chemical effect versus 70-percent cell survival (an example of the physical effect) was, in order of frequency, 500 kHz>1.0 MHz>38 kHz>2.0 MHz. Partial protection was observed for cell lysis and cell viability after sonication with 500 kHz in argon-saturated solution containing cysteamine, a free radical scavenger. These results suggest that the chemical effects of ultrasound are prominent at specific frequencies, and that free radicals induced by ultrasonic cavitation partially affect lysis and the loss of viability of rat thymocytes.

Nonsteroidal antiinflammatory drugs and a selective cyclooxygenase 2 inhibitor uncouple mitochondria in intact cells.

Uncoupling of isolated mitochondria by nonsteroidal antiinflammatory drugs (NSAIDs) has been considered relevant to the development of gastrointestinal (GI) side effects. We investigated the occurrence of NSAID-induced uncoupling of mitochondria in intact cells (rat thymocytes) compared with the effects of a selective cyclooxygenase 2 (COX-2) inhibitor. Oxygen consumption and mitochondrial membrane potential were simultaneously measured amperometrically and by distribution of radioactive tracer molecules, respectively, in the presence and absence of pharmacologically relevant concentrations of the NSAIDs indomethacin and diclofenac and the selective COX-2 inhibitor SC-236. Analysis of data by a technique related to top-down elasticity analysis permitted assessment of the influence of these compounds on individual components of cellular energy metabolism. Indomethacin, diclofenac, and SC-236 increased proton leak in isolated mitochondria. Both diclofenac and SC-236 significantly stimulated proton leak in intact cells and simultaneously inhibited substrate oxidation and ATP turnover. Oxygen consumption rates of isolated cells remained unchanged over a wide concentration range of the drugs, despite significant effects on subsystems of cellular energy metabolism. NSAIDs and selective COX-2 inhibitors have significant and equally directed effects on cellular energy metabolism. They both uncouple mitochondrial respiration and inhibit substrate oxidation and ATP turnover. However, the topical effect and selective COX-2 inhibition may not be sufficient to cause NSAID-like damage to the GI tract.

S29 ribosomal protein induces apoptosis in H520 cells and sensitizes them to chemotherapy

Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer especially in India and displays resistance to anticancer treatment. In our earlier study we had isolated a cDNA clone from rat thymocytes induced to undergo apoptosis, which was found to encode S29 ribosomal protein [Biochem. Biophys. Res. Commun. 277 (2000) 476]. In the present study an attempt has been made to find out whether enhanced expression of S29 cDNA can kill NSCLC H520 cells. We found that S29 induced apoptosis and augmented the effect of anticancer drugs. Expressions of several molecular determinants of apoptosis were analyzed in order to understand the mechanism of apoptosis induced by S29. We observed downregulation of the expression of inhibitors of apoptosis proteins (IAPs) Bcl-2, Bcl-X L, and survivin and upregulation of pro-apoptotic p53 and Bax as assessed by Western blotting. Mitochondrial release of cytochrome c and activation of initiator caspase-8 and -9 and effector caspase-3, followed by cleavage of nuclear substrate poly(ADP-ribose) polymerase, were also observed. Permeability transition as determined by changes in Δ Ψ m was not a requirement for cytochrome c release. There was a marginal increase in the release of apoptosis inducing factor (AIF) and reduction of NF-κB dependent transcriptional activity. There was non-involvement of calcium and the telomerase activity, a proliferation marker.