Thymocyte Suspensions Research Articles

Inorganic polyphosphate regulates functions of thymocytes via activation of P2X purinoreceptors

Inorganic polyphosphate (polyP) is an ancient polymer, which was proven to be a signalling molecule in the mammalian brain, mediating the communication between astrocytes via activation of P2Y1 purinoreceptors and modulating the activity of neurons. There is very limited information regarding the ability of polyP to transmit the information as an agonist of purinoreceptors in other cells and tissues. Here, we show that application of polyP to the suspension of primary thymocytes increases the concentration of intracellular calcium. PolyP evoked calcium signal was dependent on the presence of P2X inhibitors but not P2Y1 inhibitor. PolyP dependent increase in intracellular calcium concentration caused mild mitochondrial depolarization, which was dependent on inhibitors of purinoreceptors, extracellular calcium and inhibitor of mitochondrial calcium uniporter but wasn't dependent on cyclosporin A. Application of polyP modulated cell volume regulation machinery of thymocytes in calcium dependent manner. Molecular docking experiments revealed that polyP can potentially bind to several types of P2X receptors with binding energy similar to ATP - natural agonist of P2X purinoreceptors. Further molecular dynamics simulations with P2X4 showed that binding of one molecule of polyP dramatically increases permeability of this receptor-channel for water molecules. Thus, in this research we for the first time showed that polyP can interact with P2X receptors in thymocytes and modulate physiological processes.

A fast and robust protocol for immunomagnetic isolation of human CD45+ leukocytes from samples with variable start frequencies

Abstract The study of immune cell function in non-lymphoid tissue and tumors has emerged as an exciting research area, with the promise to provide novel strategies for the treatment of immune disorders, infectious diseases and cancer. A major challenge in the field is the isolation of leukocytes from tissues due to the complexity of starting samples that can be highly variable in frequency and subset composition. We describe a new 28 minute protocol optimized for the isolation of particle-free human CD45+ leukocytes from varied sample sources. Using the EasySep™ Release Human CD45 Positive Selection Kit, leukocytes are labeled with antibody complexes linked to magnetic particles and separated using an EasySep™ magnet. The isolated cells are then released from particles by resuspension in EasySep™ Release Buffer and a final magnetic separation step. Performance was assessed using human leukocytes spiked into a single cell suspension of mouse thymocytes across a range of frequencies to simulate the variable leukocyte content in tissue and tumor samples. Human CD45+ purities were 95.4 ± 4.3% from start ranges of 0.5–2.0% (n=15) and 99.1 ± 0.7% from start frequencies of ≥10% (n=18). The isolated cells are compatible with further downstream isolation and are functional, as demonstrated by T cell proliferation and IFN-gamma production assays. We have also assessed our CD45+ isolation kit from tissues of humanized NSG mice, expanding the application of our protocol to highly relevant disease and cancer models. The EasySep™ Release Human CD45 Positive Selection Kit provides a robust method for high purity isolation from diverse tissues and starting frequencies, generating particle-free cells ready for downstream evaluation.

Thymic HIV-2 infection uncovers posttranscriptional control of viral replication in human thymocytes.

A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals. This equilibrium is characterized by low to undetectable levels of viremia throughout the disease course, despite the establishment of disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 infection. Although the clinical spectrum is similar in the two infections, HIV-2 infection is associated with a much lower rate of CD4 T-cell decline and has a limited impact on the mortality of infected adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 coreceptors. We found that HIV-2 infected both thymic organ cultures and thymocyte suspensions, as attested to by the total HIV DNA and cell-associated viral mRNA levels. Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, irrespective of HIV-2 coreceptor tropism and cell differentiation stage, in agreement with the low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human thymus, but the HIV-2 replication cycle in thymocytes is impaired, providing a new model to identify therapeutic targets for viral replication control. HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to the characteristic CD4 T-cell loss. HIV-2 infection is associated with a very low rate of progression to AIDS and is therefore considered a unique naturally occurring model of attenuated HIV disease. HIV-2-infected individuals feature low to undetectable plasma viral loads, in spite of the numbers of circulating infected T cells being similar to those found in patients infected with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human thymus. We show that HIV-2 is able to infect the thymus but that the HIV-2 replication cycle in thymocytes is impaired. We propose that this system will be important to devise immunotherapies that target viral production, aiding the design of future therapeutic strategies for HIV control.

Defining cell-surface antigenic markers for mouse T and B cells.

Defining cell-surface antigenic markers for mouse T and B cells.

Thymocyte apoptosis and proliferation modeling during rat thymic involution is influenced by ovarian hormones in a thymocyte subset-specific manner

The study was aimed to define the putative role of ovarian hormones in shaping thymocyte apoptosis and proliferation during thymic involution. Thymocytes from young adult and middle-aged rats ovariectomized (Ox) before puberty were examined for apoptosis and proliferation. Apoptosis and proliferation were measured in fresh thymocyte suspensions and in their 18-hour cultures, and fresh thymocyte suspensions, respectively. The thymocyte population and the major thymocyte subsets were analyzed following triple staining using anti-CD4 and anti-CD8 monoclonal antibodies and 7-AAD to label apoptotic or proliferating cells. The frequency of apoptotic cells was lower in thymocyte suspensions and cultures from Ox rats of both ages. This reflected in a diminished frequency of apoptotic cells amongst CD4+CD8+ double positive (DP) and CD4+CD8- single positive (SP), and DP cells in young and middle-aged Ox rats, respectively. Additionally, in thymocyte cultures from Ox rats the frequency of apoptotic cells amongst CD4+CD8- and CD4-CD8+ SP cells decreased with age, but increased within DP and CD4-CD8- double negative (DN) subsets, reaching in the former subset from middle-aged Ox rats higher values than in age-matched controls. The frequency of proliferating cells was also lower in Ox rats than in controls. This reflected the lower frequency of cycling cells amongst CD4+CD8- SP and CD4-CD8+ SP thymocytes in young rats, and DP and CD4-CD8+ SP thymocytes in middle-aged rats. Besides, in both SP and DP thymocyte subsets from Ox rats the frequency of proliferating cells declined with age. In conclusion, thymocyte apoptosis and proliferation exhibit ovarian hormone-dependent thymocyte subset specific alterations during thymic involution.

Inhibition of Deoxynucleoside Kinases in Human Thymocytes Prevents dATP Accumulation and Induction of Apoptosis

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.

Inactivation of NuRD Component Mta2 Causes Abnormal T Cell Activation and Lupus-like Autoimmune Disease in Mice

Dynamic changes in chromatin structure through ATP-dependent remodeling and covalent modifications on histones play important roles in transcription regulation. Among the many chromatin modifiers identified, the NuRD (nucleosome remodeling histone deacetylase) complex is unique because it possesses both nucleosome remodeling and histone deacetylase activities. To understand the biological function of the NuRD complex, we generated a knock-out mouse model of the Mta2 (metastasis-associated protein 2) gene, which encodes a NuRD-specific component. Mta2 null mice exhibited partial embryonic lethality. The surviving mice developed lupus-like autoimmune symptoms including skin lesions, bodyweight loss, glomerulonephritis, liver inflammation, and production of autoantibodies. Transplantation of bone marrow cells from Mta2 null mice recapitulated some of the symptoms including skin lesion and bodyweight loss in the recipient mice. Mta2 null T lymphocytes showed normal development but hyperproliferation upon stimulation, which correlates with hyperinduction of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma. T cell hyperproliferation, but not other autoimmune symptoms, was observed in T cell-specific Mta2 knock-out mice. Mta2 null T cells produced more IL-4 and IFN-gamma under Th2 activation conditions, but normal levels of IL-4 and IFN-gamma under Th1 activation conditions. Furthermore, we found that IL-4 is a direct target gene of Mta2. Our study suggests that Mta2/NuRD is involved in modulating IL-4 and IFN-gamma expression in T cell immune responses, and gene expression in non-T cells plays an important role in controlling autoimmunity.

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.

Control of Autoimmune Myocarditis and Multiorgan Inflammation by Glucocorticoid-Induced TNF Receptor Family-Related Proteinhigh, Foxp3-Expressing CD25+ and CD25− Regulatory T Cells

Anomalies of naturally occurring CD4+ regulatory T cells (Treg) cause severe autoimmune/inflammatory diseases in humans and rodents. The transcription factor Foxp3 is currently the most specific marker for natural CD4+ Treg, but it would be useful if other Treg markers, particularly cell surface molecules, could be elucidated. We demonstrate in this study that the vast majority of Foxp3-expressing CD4+ T cells (whether CD25+ or CD25-) show constitutive high-level expression of glucocorticoid-induced TNFR family-related gene/protein (GITR). Transfer of T cell or thymocyte suspensions depleted of GITR(high) cells produces in BALB/c nude mice a wider spectrum and more severe forms of autoimmune diseases than does transfer of similar cell suspensions depleted of CD25+ CD4+ T cells only. Notably, mice that receive cells depleted of GITR(high) populations develop severe multiorgan inflammation that includes fatal autoimmune myocarditis resembling giant cell myocarditis in humans, accompanying high-titer anti-myosin autoantibodies. Similar transfer of GITR(high)-depleted cells from prediabetic NOD mice to NOD-SCID mice accelerates the development of diabetes and induces skeletal muscle myositis and other autoimmune/inflammatory diseases. We conclude that GITR(high), Foxp3-expressing natural Treg, containing both CD25+ and CD25- cell populations, contribute to preventing a variety of autoimmune/inflammatory diseases, and depletion of these cells allows the activation of even weak or rare autoreactive T cells yielding widespread severe autoimmune disease. Diseases induced in this way include many which have been suspected of an autoimmune etiology in humans without much evidence. GITR(high), Foxp3-expressing natural Treg represent a potential target for the treatment and prevention of these diseases.

Diethylstilbestrol (DES)-Induced Fetal Thymic Atrophy in C57BL/6 Mice: Inhibited Thymocyte Differentiation and Increased Apoptotic Cell Death

Treatment of pregnant C57Bl/6 mice with 48 mu g/kg diethylstilbestrol (DES) on gestation days (GDs) 14 and 16 resulted in both decreased day 18 fetal thymic cellularity as well as alterations in thymocyte phenotype. Histopathologic examination of GD 18 fetal thymi from DES-exposed dams demonstrated a decrease in thymic size and cellularity and an increase in pyknotic nuclei, indicative of apoptosis, relative to control thymi. Thymic architecture was also altered by DES treatment with a decrease in the distinction between the cortical and medullary regions. Flow cytometric staining of day 18 thymocyte suspensions with the apoptotic marker 7-aminoactinomycin D showed a decrease in thymocyte viability after DES, and a concomitant increase of thymocytes in early apoptosis. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, trends toward increased apoptosis were present in the CD4+CD8+ and CD4+CD8- subpopulations, and significantly increased apoptosis occurred in the CD4-CD8- and CD4-CD8+ subpopulations. These histopathologic and flow cytometric findings support enhanced apoptosis of thymocytes as a contributing factor to fetal thymic atrophy caused by DES.

Tetrachlorodibenzo-p-Dioxin (TCDD) Inhibits Differentiation and Increases Apoptotic Cell Death of Precursor T-Cells in the Fetal Mouse Thymus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy as well as alterations in thymocyte maturity in mice. Multiple mechanisms for thymic hypocellularity have been suggested, and include an increase in thymocyte apoptosis, a maturation arrest of thymocyte development, inhibited thymocyte proliferation, and a diminution of seeding of the thymus by the hematopoietic progenitors in the fetal liver or adult bone marrow. Fetal mice are highly sensitive to hypocellularity induction by TCDD when the chemical is administered during the window of thymic development, between days 10 and 18 of gestation. Treatment of pregnant C57Bl/6 mice in the present experiments with doses of 5 or 10 μ g/kg TCDD by oral gavage on gestation days 14 and 16 severely depressed day 18 thymic cellularity. Histopathologic evaluation of day 18 fetal thymi showed disruption of the normal organ architecture with loss of clear distinction between cortical and medullary regions after TCDD. A decrease in thymocyte density was noted in all regions, and was most dramatic in the cortical zones where pyknotic cells were increased by TCDD treatment. Using day 18 thymocyte suspensions and flow cytometry, the marker 7-AAD showed a decrease in viable thymocytes from TCDD-treated fetal mice, and a concomitant and dose-related increase of thymocytes in early apoptosis. Specifically, relative to control, thymocytes from the 5 and 10 μg/kg TCDD exposure groups displayed 1.9% and 5.3% respective increases in early apoptotic cells. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, the enhanced apoptosis occurred in the CD4+CD8+ phenotype with no significant apoptosis seen in the CD4−CD8−, CD4+CD8−, or CD4−CD8+ thymocytes. Given the rapid clearance of apoptotic cells from the thymus, these histopathologic and cytometric data suggest increased thymocyte apoptosis contributes to fetal thymic atrophy after TCDD exposure.

Differential expression of notch signaling-related transcripts accompanies pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures.

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.

Effect of X-Ray and UV irradiation of the C 60 fullerene aqueous solution on biological samples

The dependence of the optical absorption density on the UV irradiation time of a biological sample (the suspension of thymocytes, isolated from Wistar rat thymus) and the DNA structural state in cells under X-ray irradiation in the presence of the C 60 fullerene aqueous solution were investigated and analyzed in detail. The significant pro-oxidant effect of this material at the low concentration of fullerene C 60 (10 −5 M) was revealed.

Modification of thymocytes membrane radiooxidative damage and apoptosis by eugenol.

Radiation-induced membrane oxidative damage, generation of reactive oxygen species (ROS), and cellular apoptosis, as well as their modification by a natural antioxidant from plant source, eugenol, have been investigated in mouse thymocytes. Thymocyte suspension was irradiated by gamma-rays, and the malondialdehyde (MDA) formation was measured with the thiobarbituric acid reactive species (TBARS) method. The results showed an increase in MDA in irradiated (2 Gy) thymocytes, which was inhibited in samples treated with increasing concentrations of eugenol (10-200 microM) prior to irradiation. The concentration of eugenol required to inhibit half of the MDA formation (IC(50)) in irradiated thymocytes was 100 microM. A dose-dependent increase in the generation of ROS was observed in irradiated thymocytes (0.5-200 cGy) as measured by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA), which was, however, inhibited by eugenol administered before irradiation. Furthermore, experiments showed a significant decrease in thymocytes apoptosis by antioxidant as measured by annexin-V labeling protocol. The inhibition of apoptosis by membrane-localized antioxidants such as eugenol, isoeugenol, and alpha-tocopherol acetate was more effective than a cytosolic antioxidant such as ascorbic acid. The results suggest an effective prevention of membrane and cellular damage in irradiated thymocytes by eugenol. It is inferred that damage to membrane played a significant role in radiation-induced cellular apoptotic death, which was markedly modified by membrane-specific antioxidants.

High Incidence of Thymic Epithelial Tumors in E2F2 Transgenic Mice

In virtually all human tumors, genetic and epigenetic alterations have been found which affect the INK4/-CYCLIN D/RB pathway, which regulates cell cycle entry and exit in normal cells. E2F transcription factors are important downstream components of this pathway, which act by controlling the expression of genes involved in DNA replication and cell cycle progression. To determine whether E2F2 deregulation promotes proliferation and tumorigenesis in vivo, we generated E2F2 transgenic mice, in which the Emu and murine pim1 promoter (pp) direct high expression of E2F2 in thymic epithelial cells. Emu-pp-E2F2 mice start to develop cytokeratin- and ER-TR4-positive cortical thymomas from the age of 20 weeks, and within 1 year, nearly all mice succumb to gross thymic epithelial tumors. General thymic morphology is largely maintained, but T cell development is perturbed in thymomas, with proportionately less CD4(+)CD8(+) double-positive thymocytes. In the first 3 months, E2F2 transgenic thymi exhibit only mild epithelial hyperplasia, and thereafter thymomas arise stochastically, probably following additional mutations. Interestingly, Emu-pp-E2F1 mice do not display cortical thymomas. These data argue that E2F2 promotes unscheduled cell division and oncogenic transformation of thymic epithelial cells.

Spectral analysis of the Ca 2+ concentration measurements in living cells

The effects of the experimental conditions and protein microenvironment on the fluorescence spectra of the Ca 2+-sensitive dye indo-1 in solutions and suspension of thymocytes have been studied. The temperature dependence of the contributions of L-, LM- and LP-forms of the dye in the integral fluorescent spectrum of indo-1 were estimated. The changes of Ca 2+ concentration in thymocytes of the rat during activation evoked by high KCl solution were reported.

Functional Uncoupling of T-cell Receptor Engagement and Lck Activation in Anergic Human Thymic CD4+T Cells

Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human thymic CD1a-CD4+ T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505. Confocal microscopy demonstrated that colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.

Activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) on undernourished and renourished rats’ thymus

We studied the effect of administration of a low quality dietary protein, from weaning onwards, on the thymus of undernourished rats and the posterior effect of refeeding with a high quality dietary protein. Changes in thymus weight and the activity of Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP) on thymus, were determined. Wistar rats were suckled in groups of 14–16 per dam since birth to weaning (23 days) to obtain undernutrition. At weaning, a group of 14–16 rats received pre-cooked maize flour (Protein content: 6.5%) for 18 days. One group was sacrificed (M) and the other rats were refed with the casein diet (Protein content: 20%) during 20 days (R). The age-matched control groups were fed stock diet since 40 (C40) and 60 (C60) days of age, respectively. At the end of the experimental period, body (Bw) and thymus weight were determined. ADA and PNP activities were determined in thymocyte suspensions. Highly significant differences in thymus weight—expressed as mg or mg/Bw 0.75—and the activity of ADA and PNP were observed in rats fed the experimental diet containing maize flour, when compared to the respective age-matched control. No statistical differences were observed between R and C60.The administration of a high quality dietary protein to undernourished weanling rats is capable to reverse the damage produced by the low quality dietary protein on thymus weight and ADA and PNP thymus activities.

Les cellules dendritiques thymiques humaines

Human thymic dendritic cells (DC) were enriched from thymocyte suspension by low density fractionation and elimination of CD3, CD8, CD19, CD56 and CD34-positive cells. Flow cytometry analysis shows that they belong to two distinct populations. The prominant (2/3) one is CD4+ CD45RA+ CD33-CD11c-, mostly immature as it lacks CD80, CD83 and CD86, and is HLA-DRint. High expression of CD123 and expression of pre-TCR-alpha transcripts links this subset to lymphoid, Interferon-producing cells. The other DC are typically CD4+ CD33+ CD11c+ myeloid cells mostly mature CD83+ HLA-DRhi. Both subsets could be generated in vitro from thymic CD34+ progenitors. Finally although experimental evidence ascribed to thymic DC a mjor role in establishing central tolerance through deletion of self reactive thymocytes, the respective fonctions of human lymphoid and myeloid subsets in the human thymus remain to be established.

Nitric Oxide Production and Mitochondrial Dysfunction during Rat Thymocyte Apoptosis

Production of nitric oxide (NO) by mitochondrial membranes as methemoglobin formation sensitive to N(G)-methyl-l-arginine inhibition and mitochondrial O(2) consumption in metabolic states 3 and 4 and the respiratory control (state 3/state 4) were measured at early stages of rat thymocyte apoptosis. Programmed cell death was induced by addition of methylprednisolone and etoposide to thymocyte suspensions. Increased NO production by mitochondrial membranes was observed after 30 min of methylprednisolone and etoposide addition and was accompanied by mitochondrial respiratory impairment as an early phenomenon in apoptotic thymocytes. The respiratory control in isolated mitochondria from untreated thymocytes was 4.2 +/- 0.2 and decreased to 3.1 +/- 0.2 and 1.9 +/- 0.3 after 1 h of methylprednisolone and etoposide treatment, respectively. The low mitochondrial respiratory control was accompanied by a marked decrease in GSH and cytochrome c content. Moreover, an inhibitory effect in the amount of apoptosis due to thymocyte pretreatment with N(G)-methyl-l-arginine and N(omega)-nitro-(l)-arginine (l-NNA), indicate that nitric oxide production is closely involved in the signaling of rat thymocyte apoptosis.