Thymocyte Membrane Research Articles

Semaphorin-3A Inhibits Proliferation, but Does Not Affect Apoptosis of Mouse Thymocytes In Vitro.

Neuronal factor semaphorin-3A is viewed as immune suppressant of peripheral T lymphocytes, but it can also negatively affect activity of the thymus, the central organ of the immune system. The study examined the effects of this factor on proliferative activity and apoptosis of mouse thymocytes in vitro. Semaphorin-3A inhibited spontaneous and mitogen-stimulated proliferative activity of thymocytes producing no effect on the development of apoptosis in these cells. Flow cytometry revealed expression of semaphorin-3A receptors neuropilin-1 and plexin-A1 on thymocyte membranes. Approximately 13% thymocytes simultaneously expressed both receptors. The study suggests that semaphorin-3A, which is constitutively synthesized in thymic stroma in vivo, can play the role of inhibitory factor during thymocyte maturation.

Membrane permeability changes in radiation-induced apoptotic mouse thymocytes

Objective: Radiation-induced alterations in permeability of plasma membrane and associated apoptotic changes were investigated in apoptotic thymocytes induced by radiation. Materials and Methods: Immature mouse thymocytes suspended in RPMI 1640 were irradiated by γ-rays for desired doses up to 10 Gy and these cells were examined after incubation (up to 24 h) at 37°C. Radiation-induced changes in plasma membrane of thymocytes were determined by fluorescence technique using fluorescein diacetate (FDA) and trypan blue (TB) method. Induction of apoptosis in irradiated cells was determined using annexin-V as fluorescence probe method and measurement of cytosolic caspase-3 activity. Results: Radiation-induced apoptotic thymocytes showed an increase in membrane permeability as observed by leakage of FDA, which was poorly detected by TB. FDA could sensitively detect the dose-dependent variation in membrane permeability alterations in the range of 0.5–2 Gy incubated at 37°C. In radiation-induced apoptotic death, externalization of phosphatidylserine is an early event than caspase-3 activation. On incubation of cells, an increase in caspase-3 activity and proportion of annexin-positive cells was observed, which further increased after irradiation. Conclusion: Our results showed membrane permeability changes induced by γ-irradiation, which seem to be closely associated with the apoptosis in thymocytes.

ATP Binding Cassette Transporter ABCA7 Regulates NKT Cell Development and Function by Controlling CD1d Expression and Lipid Raft Content

ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7−/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function.

Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures.

The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4-CD8- double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells' influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes.

New inhibitors of glucosylceramide synthase and their effect on cell fate

Abstract Glucosylceramide (GlcCer) is an essential glycosylated lipid found in organisms ranging from fungi to mammals. It is composed of a hydrophilic β-linked glucose and a hydrophobic ceramide, with a predominant content of sphingosine in mammals (d18:1). GlcCer is the precursor of a large scale of different glycosphingolipids. This cerebrozide is synthesized from uridine diphosphate-glucose and ceramide by a GlcCer synthase (UDP-glucose:ceramide glucosyltransferase; UGCG, EC 2.4.1.80). GlcCer-based sphingolipids have been identified as important mediators of a variety of cellular functions and their disequilibrium leads to pathological process development and may induce several diseases progression. Therefore, design of UGCG inhibitor represents an important topic for pharmaceutical research. In this paper, we aimed to study effects of newly synthesized derivatives of (±)-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, known UGCG inhibitor) on: i) activity of UGCG in vitro; ii) thymocytes viability; iii) calcium transport through plasma membrane of thymocytes; iv) induction of apoptosis and autophagy in thymocytes. Thymocytes were isolated from thymus of three to seven weeks old mice (ICR strain). The key factors influencing the effect of PPMP analogues were their concentration, chemical structure and incubation time. Derivatives were able to change Ca2+ transport already after 15 min of cultivation, but their effects on cell viability were manifested at least after 12 h of cultivation. Four from fifteen studied compounds affected UGCG activity after four hour lasting cultivation, - but without correlation with data relating to effects on calcium transport and/or cell viability. Most potent UGCG inhibitor was chosen and applied for induction of apoptosis and autophagy in thymocytes. This inhibitor induced typical DNA fragmentation and upregulation of LC3B protein as autophagy marker, after 2 h and 4 h cultivation, respectively.

Expression and distribution of the adrenomedullin system in newborn human thymus.

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.

Is there a Possible Single Mediator in Modulating Neuroendocrine–thymus Interaction in Ageing?

The restoration of the thymic functions and the thymic re-growth may be achieved in old mice by some endocrinological (melatonin) or nutritional interventions (arginine or zinc), suggesting that the thymic involution in old age is a phenomenon secondary to age-related alterations occurring in neuroendocrine-thymus interactions. The targets for the thymic restoration may be hormone receptors and cytokines, strictly related to the presence of two nutritional factors, such as arginine and zinc, which are in turn essential for the efficiency of neuroendocrine-immune network both in ontogeny and ageing. The effect of melatonin is largely due to the presence of its specific receptors on cell membrane of thymocytes and Thymic Epithelial Cells (TECs). TECs synthesize thymulin peptide that is required for T-cell differentiation and maturation within the thymus gland. In this context, the role of zinc is pivotal because it is involved, through "zinc finger motifs", in the gene expression of melatonin receptors, in cell proliferation, apoptosis and thymulin reactivation. Zinc is also required for the biological action of arginine, via Nitric Oxide pathway. Therefore, the beneficial effect of melatonin or arginine on neuroendocrine-thymus interaction in ageing can also occur via a better zinc pool redistribution within the body where the capability of the zinc-binding proteins Metallothioneins (MT) in zinc release has a key role. These findings suggest that zinc, via MT buffering, can be a single mediator in modulating neuroendocrine-thymus interaction in ageing.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal–isobaric conditions (310.15K and 1atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes.

Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (gamma-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [3H]bestim binds with a high affinity to murine peritoneal macrophages (Kd 2.1 +/- 0.1 nM) and thymocytes (Kd 3.1 +/- 0.2 nM), as well as with plasma membranes isolated from these cells (Kd 18.6 +/- 0.2 and 16.7 +/- 0.3 nM, respectively). The specific binding of [3H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (Ki 0.9 +/- 0.1 and 1.1 +/- 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [3H]bestim. Bestim in the concentration range of 10(-10) to 10(-6) M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.

Stimulating effect of erythropoietin on thymocyte energetics established in vitro with a potential-sensitive fluorescent probe

Effects of a peptide hormone—human recombinant erythropoietin (EPO)—on transmembrane potential (TMP) and a number of active mitochondria in rat thymocytes were studied in vitro using the fluorescent cationic probe 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). It was established that EPO changes electric potentials on the surface of cellular membranes of rat thymocytes. Changes in fluorescent signals of DSM were found to depend on the EPO concentration and physiological status of thymus cells. EPO concentrations sufficient to increase average fluorescence intensity of DSM ( $$\tilde F$$ ) in thymus cell mitochondria were established in vitro. These effects were stimulated by increasing the average number of energized mitochondria (N m ) able to accumulate the fluorescent cationic probe. These changes can also be due to elevation of proton potential on the mitochondrial membrane and/or of electric potential of the plasma membrane. Experimental values of positive correlation coefficients between mean values of $$\tilde F$$ and N m in experimental (EPO) thymus cell samples differed from those in control. In the presence of EPO, $$\tilde F$$ increased nonlinearly with N m due to different responsiveness of thymocytes to EPO and change in polarization of the outer mitochondrial membrane in some EPO-stimulated cells. In the majority of cases, the peak in the distribution histogram of $$\tilde F$$ was shifted towards augmentation of the DSM signal. The EPO response of cells isolated from different thymuses depended on the initial level of the average fluorescent signal of DSM in mitochondria, which testifies to differences in physiological statuses of animal thymuses and experimental animals at large.

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [3H]bestim was found to bind with high affinity to mouse peritoneal macrophages (Kd 2.1 ± 0.1 nM) and thymocytes (Kd 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (Kd 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [3H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (Ki 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [3H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane

Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.

The potential role of caveolin‐1 in inhibition of aquaporins during the AVD

During apoptosis, the first morphological change is a distinct cell shrinkage known as the AVD (apoptotic volume decrease). This event is driven by a loss of intracellular K(+), which creates an osmotic gradient, drawing water out of the cell through AQPs (aquaporins). Loss of water in balance with K(+) would create a shrunken cell with an equivalent intracellular concentration of K(+) ([K(+)](i) = 140 mM). However, we have previously shown that the [K(+)](i) of the shrunken apoptotic cell is 35 mM, and this level is absolutely essential for the activation of apoptotic enzymes. We have recently found that AQPs are inactivated following the AVD, so that continued loss of K(+) will reduce the intracellular concentration to this critical level. Using thymocytes, we have investigated the expression profile and regulation of the AQP family members. In the present study, we have found that AQP1, AQP8 and AQP9 are present in non-apoptotic thymocytes and localized primarily to the plasma membrane. Expression and localization did not change when these cells were induced to undergo apoptosis by growth factor withdrawal for 24 h. To explore other possible mechanisms by which these water channels are inactivated, we investigated their association with CAV-1 (caveolin-1), binding to which is known to inactivate a variety of proteins. We found that CAV-1 is present in thymocytes and that this protein co-localizes with a portion of AQP1 in normal (non-apoptotic) thymocytes. However, thymocytes induced to undergo apoptosis greatly increase their AQP1/CAV-1 association. Taken together, these results indicate that AQPs are localized to the plasma membrane of shrunken apoptotic thymocytes where increased binding to CAV-1 potentially inactivates them. AQP inactivation, coupled with continued K(+) efflux, then allows the [K(+)](i) to decrease to levels conducive for the activation of downstream apoptotic enzymes and the completion of the apoptotic cascade.

Prolactin induced reversal of glucocorticoid mediated apoptosis of immature cortical thymocytes is abrogated by induction of tumor

Glucocorticoid (GC) and prolactin (PRL) are the two neuroendocrines that regulate thymocyte differentiation and maintain the immune homeostasis during stress. We found that dexamethasone (Dex), a synthetic GC, induced apoptosis in normal immature cortical thymocytes which remained unaltered in the presence of Elrich's ascitic carcinoma (EAC). PRL protected the normal CD4 + CD8 + cortical thymocytes from Dex-induced apoptosis but failed to alter the effect of Dex in tumor-bearing mice. Dex-treated normal thymocytes became unresponsive to PRL in presence of tumor cell culture supernatant. Low binding affinity of the microsomal membranes of thymocytes to PRL and absence of the mRNA of a particular form of prolactin receptor (PRL-R) suggest the presence of a different PRL-R in CD4 + CD8 + thymocytes of EAC-bearing mice. The induction of tumor may alter the PRL-R that can be correlated with the failure of PRL in rescuing CD4 + CD8 + immature cortical thymocytes from GC induced death.

T-cell receptor (TCR)/CD3 is involved in sulfated polymannuroguluronate (SPMG)-induced T lymphocyte activation

Sulfated polymannuroguluronate (SPMG) has entered the Phase II clinical trial as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate in China. Proliferation assays showed that SPMG was effective at enhancing the proliferative response of T lymphocytes either with or without concanavalin A (ConA) stimulation. Flow cytometry (FCM) and fluorescence microscope examination revealed the significant binding of SPMG to T lymphocytes. The significant engagement of SPMG with TCR/CD3 complex was verified by competitive inhibition assay and one of the SPMG binding proteins purified by affinity chromatography from thymocyte membrane preparations was further confirmed to be CD3 component of TCR/CD3 complex via Western blotting analysis. In addition, SPMG was demonstrated to dramatically interact with ConA in a multivalent manner by surface plasmon resonance (SPR) assay. Notably, the concomitant presence of ConA and SPMG facilitated each other's binding to T cells. Together, the simultaneous interactions of SPMG with TCR/CD3 and with ConA can be highly proposed to facilitate the cross-linking of these molecules, and thus favoring costimulatory signaling, which serves to well explain the immunopotentiation and anti-human immunodeficiency virus (HIV) activities of SPMG.

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.

Glucocorticoid agonistic and antagonistic effects of mifepristone and onapristone on thymocyte subset composition and CD26/dipeptidyl peptidase IV activity in infant male rats

Antiglucocorticoid activities of two antigestagens-antiglucocorticoids (AGs)—mifepristone and onapristone—were tested in hydrocortisone-treated suckling male rats. Hydrocortisone (HC) treatment in vivo resulted in (1) reduction of the relative thymus weight and absolute thymocyte counts; (2) relative decrease of the CD4 +CD8 + thymocyte proportion accompanied by an increase of single-positive and double negative thymocyte populations, the latter of which contained large CD3-negative cells expressing a high level of CD26 on their surface; (3) increase of specific dipeptidyl peptidase IV (DPP IV) activity in thymocyte homogenates. Both AGs suppressed the systems (1) and (2) to a comparable extent. When administered alone, mifepristone and onapristone at higher doses exhibited a slight thymolytic effect as revealed by the reduction of the relative thymus weight and thymocyte counts, accompanied by some reduction of the numbers of cycling thymocytes. These effects were limited to the early postnatal period (days 12–17). A comparable agonistic effect of AGs was not observed in systems (2) and (3). Neither HC nor AGs influenced the sialylation pattern of thymocyte membrane bound CD26/DPP IV, which was exclusively of α2,6-type, as demonstrated by analytical isoelectric focusing (IEF) and PAGE analysis in combination with the application of neuraminidases, specific lectins and histochemical staining for DPP IV activity in the gels.

In vitro studies on radiation induced membrane oxidative damage in apoptotic death of mouse thymocytes

Radiation oxidative damage to the plasma membrane of cells and its consequences in the mechanism of apoptotic death have been receiving growing attention of radiation scientists in recent years. We have employed a fluorescence probe method to determine changes in the permeability and fluidity of the plasma membrane of mouse thymocytes following γ irradiation mwithin a low to moderate radiation dose range (a few cGy to 10 Gy;·0.71 Gy/min). In vitro studies have shown that radiation induced membrane changes were correlated with the induction of apoptotic cell death. Studies have also been carried out to evaluate the generation of intracellular reactive oxygen species (ROS) in response to radio-oxidative damage to thymocytes using a cytosolic fluorescence probe, 2'-7'- dichlorodihydrofluorescien diacetate (DCH-FDA). The apoptosis of the thymocytes was determined by measuring nuclear condensation using propidium iodide (PI) and DNA fragmentation by gel electrophoresis. The viability of the thymocytes was determined by the trypan blue exclusion method which was found to gradually decrease after incubation of irradiated cells for 7 h. Thymocytes labeled with DCH-FDA in PBS at 25°C showed remarkably increased fluorescence intensity, measured after 30 min of irradiation (10 Gy), indicating an enhanced formation of intracellular reactive oxygen species. The DCH-FDA probe was able to sensitively detect irradiation effects on cells at lower doses. The population of thymocytes with reduced nuclear diameter was found to progressively increase with the postirradiation incubation time, following exposure to a particular radiation dose. Moreover, fragmentation of nuclear DNA in irradiated thymocytes, as observed by the pattern of ladder formation on agarose gel, was found to significantly increase on incubations beyond 3 h at 37°C. Results indicate that the enhanced amount of intracellular reactive oxygen species generated in response to radiooxidative stress were linked with the induction of γ irradiation of apoptotic death in thymocytes. These observations and a highlight of emerging scenario in the area of low dose radiation effects at cellular and membrane levels are described.

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes.

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.

Influx of Calcium through a Redox-sensitive Plasma Membrane Channel in Thymocytes Causes Early Necrotic Cell Death Induced by the Epipolythiodioxopiperazine Toxins

Gliotoxin, a member of the epipolythiodioxopiperazine (ETP) class of toxins, induces both apoptotic and necrotic cell death in a concentration-dependent manner. Whereas the specific trigger for apoptotic death caused by these toxins is unclear, the reactive disulfide bond in the ETP toxins is required for biological activity. Thus it is likely that it is the interaction of this disulfide moiety with macromolecules in cells that was responsible for activity of ETP toxins. Here we present evidence that necrosis induced by gliotoxin and a simple synthetic ETP toxin is largely because of an influx of extracellular calcium through a redox-sensitive calcium channel in the plasma membrane of murine thymocytes. The calcium rises are strongly dependent on the pH of the external medium and the presence of external calcium and are abrogated and/or reversed by the presence of dithiothreitol, cell impermeant glutathione, and the calcium channel blocker Ni(2+). Comparisons with thapsigargin, which indirectly causes release of calcium from internal stores, indicates that ETP toxins do not provoke calcium rises by store depletion. A mechanism of oxidation by ETP toxins of cell surface thiol groups resulting in direct entry of calcium through a redox active channel in the plasma membrane is proposed. Necrotic but not apoptotic cell death was abrogated by inhibition of calcium entry.