Dexamethasone-induced Apoptosis In Thymocytes Research Articles

Efferocytosis-induced lactate enables the proliferation of pro-resolving macrophages to mediate tissue repair.

The clearance of apoptotic cells by macrophages (efferocytosis) prevents necrosis and inflammation and activates pro-resolving pathways, including continual efferocytosis. A key resolution process in vivo is efferocytosis-induced macrophage proliferation (EIMP), in which apoptotic cell-derived nucleotides trigger Myc-mediated proliferation of pro-resolving macrophages. Here we show that EIMP requires a second input that is integrated with cellular metabolism, notably efferocytosis-induced lactate production. Lactate signalling via GPR132 promotes Myc protein stabilization and subsequent macrophage proliferation. This mechanism is validated in vivo using a mouse model of dexamethasone-induced thymocyte apoptosis, which elevates apoptotic cell burden and requires efferocytosis to prevent inflammation and necrosis. Thus, EIMP, a key process in tissue resolution, requires inputs from two independent processes: a signalling pathway induced by apoptotic cell-derived nucleotides and a cellular metabolism pathway involving lactate production. These findings illustrate how seemingly distinct pathways in efferocytosing macrophages are integrated to carry out a key process in tissue resolution.

PFKFB2-mediated glycolysis promotes lactate-driven continual efferocytosis by macrophages.

Resolving-type macrophages prevent chronic inflammation by clearing apoptotic cells through efferocytosis. These macrophages are thought to rely mainly on oxidative phosphorylation, but emerging evidence suggests a possible link between efferocytosis and glycolysis. To gain further insight into this issue, we investigated molecular-cellular mechanisms involved in efferocytosis-induced macrophage glycolysis and its consequences. We found that efferocytosis promotes a transient increase in macrophage glycolysis that is dependent on rapid activation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), which distinguishes this process from glycolysis in pro-inflammatory macrophages. Mice transplanted with activation-defective PFKFB2 bone marrow and then subjected to dexamethasone-induced thymocyte apoptosis exhibit impaired thymic efferocytosis, increased thymic necrosis, and lower expression of the efferocytosis receptors MerTK and LRP1 on thymic macrophages compared with wild-type control mice. In vitro mechanistic studies revealed that glycolysis stimulated by the uptake of a first apoptotic cell promotes continual efferocytosis through lactate-mediated upregulation of MerTK and LRP1. Thus, efferocytosis-induced macrophage glycolysis represents a unique metabolic process that sustains continual efferocytosis in a lactate-dependent manner. The differentiation of this process from inflammatory macrophage glycolysis raises the possibility that it could be therapeutically enhanced to promote efferocytosis and resolution in chronic inflammatory diseases.

Efferocytosis induces macrophage proliferation to help resolve tissue injury

Apoptotic cell clearance by macrophages (efferocytosis) promotes resolution signaling pathways, which can be triggered by molecules derived from the phagolysosomal degradation of apoptotic cells. We show here that nucleotides derived from the hydrolysis of apoptotic cell DNA by phagolysosomal DNase2a activate a DNA-PKcs-mTORC2/Rictor pathway that increases Myc to promote non-inflammatory macrophage proliferation. Efferocytosis-induced proliferation expands the pool of resolving macrophages invitro and in mice, including zymosan-induced peritonitis, dexamethasone-induced thymocyte apoptosis, and atherosclerosis regression. In the dexamethasone-thymus model, hematopoietic Rictor deletion blocked efferocytosing macrophage proliferation, apoptotic cell clearance, and tissue resolution. In atherosclerosis regression, silencing macrophage Rictor or DNase2a blocked efferocyte proliferation, apoptotic cell clearance, and plaque stabilization. In view of previous work showing that other types of apoptotic cell cargo can promote resolution in individual efferocytosing macrophages, the findings here suggest that signaling-triggered apoptotic cell-derived nucleotides can amplify this benefit by increasing the number of these macrophages.

Mixed Lineage Kinase 3 Promotes Dexamethasone-Induced Thymocyte Apoptosis

Abstract Dexamethasone (DEX) is a corticosteroid known to regulate T-lymphocyte growth/differentiation and used in the clinic as an anti-inflammatory and immunosuppressant agent. The glucocorticoid hormones, including DEX, can induce thymocyte apoptosis. It has been reported that DEX-induced thymocyte apoptosis is associated with an early stage ceramide generation. Since we reported earlier that ceramide induces Mixed Lineage Kinase 3 (MLK3) activation and therefore, we probed whether MLK3 inhibition could attenuate DEX-induced thymocyte apoptosis. Here, we report that genetic loss of MLK3, a member of MAP3K family attenuates DEX-induced apoptosis in thymocytes. To understand any differences in thymic T cell surface markers at the basal level between WT and MLK3−/− mice, we determined protein expression of various surface proteins involved in T cell activation and apoptosis. Flow cytometry analyses indicated that the genetic loss of MLK3 increases CD27 expression on thymic CD4+, CD8+, and CD4+CD8+ T cells; however, CD28 expression was mainly increased on CD8+ and CD4+CD8+ T cells due to loss of MLK3. Interestingly, genetic loss of MLK3 paradoxically regulated CD70 expression on thymic and splenic T cells. Furthermore, 4–5 weeks old WT and MLK3−/− mice were challenged with DEX for 24 h, and apoptosis in thymic T cells was determined. The flow cytometry results showed a significant decrease in the DEX-induced apoptosis in thymic CD4+, CD8+, and CD4+CD8+ T cells, isolated from MLK3−/− mice. Taken together, these results suggest that MLK3 could be a potential target for DEX-induced thymocyte apoptosis and its inhibitors can be used to mitigate thymocyte apoptosis.

In vivo protective effect of betulinic acid on dexamethasone induced thymocyte apoptosis by reducing oxidative stress.

Dexamethasone (Dex), a synthetic glucocorticoid, is strictly controlled for use due to its serious side effects, including immune suppression. Betulinic acid (BA), an antioxidant prepared from the white birch, exhibits immunomodulation properties. To assess the implications and investigate the mechanisms of BA-elicited immunomodulation, we hypothesized that Dex induced thymocyte apoptosis via oxidative stress could be lessened by BA. Mice were given oral doses of BA (0.25, 0.5, and 1.0mg/kg) daily for 14 days, and induced oxidative stress by giving a single dose of Dex intraperitoneal at the dosage of 25mg/kg body weight 8h after the last administration of BA. Dex administration alone significantly decreased antioxidant enzyme activities, while significantly increased reactive oxygen species (ROS) production, lipid peroxidation, mitochondrial dysfunctions, caspase-3 activation and cellular apoptosis. However, pretreatment with BA dose-dependently mitigated Dex-induced oxidative damage after 14 days of feeding. In addition to ROS scavenging activity in Dex-induced thymocyte, BA administration decreased lipid peroxidation, up-regulated antioxidant enzymes, restored mitochondrial function, increased Bcl-2 expression but reduced Bax expression, inhibited caspase-3 activation, and improved cell survival. These findings reveal a protective capability of BA against Dex-induced cell death by reducing oxidative stress via mitochondrial mediated signal pathway which could be the potential mechanism underlying BA-elicited immunomodulation.

Involvement of cPLA2Inhibition in Dexamethasone-Induced Thymocyte Apoptosis

Various molecular mechanisms have been suggested to be involved in dexamethasone induced thymocyte apoptosis. In this study we show that pharmacological inhibition of cytoplasmic PLA2 in mouse thymocytes for 18 h with arachidonyl trifluoromethyl ketone (AACOCF3) (10 microM) and palmitoyl trifluoromethyl ketone (PACOCF3) (10 microM) induced a drastic increase of thymocyte apoptosis comparable to that observed following Dex (10(-7) M) treatment, while inhibition of secretory PLA2 with p-bromophenacyl bromide (pBPB) (20 microM) did not. AACOCF3-induced thymocyte apoptosis, similarly to Dex-induced thymocyte apoptosis, was eliminated by cell pre-treatment with the PI-PLCbeta inhibitor, U73122, but not by the PC-PLC inhibitor D609. These observations were corroborated by the ability of AACOCF3, like Dex, to induce a rapid and transient increase in DAG generation. In addition, AACOCF3-induced apoptosis involved the activation of the acidic sphingomyelinase (aSMase) but not of the neutral sphingomyelinase (nSMase), as evaluated by measurements of enzyme activity in cell extracts following thymocyte exposure to AACOCF3 and by the ability of monensin to inhibit AACOCF3-induced thymocyte apoptosis. In addition, the AACOCF3 apoptotic effect resulted in an early increase of ceramide levels. AACOCF3-induced thymocyte apoptosis involved the activation of caspase 3, and cell pre-treatment with a caspase 3 inhibitor prevented AACOCF3-induced apoptosis. These observations suggest that cPLA2 inhibition may have a role in Dex-induced thymocyte apoptosis and highlight the importance of cPLA2 activity in thymocyte survival.

Tactivin in the regulation of dexamethasone-induced apoptosis in thymocytes

The level of natural apoptosis in rat thymus on day 18 of embryo development attained 25%, while at subsequent terms it was about 5%. In the spleen, this parameter gradually decreased from 15 to 37% starting from day 18 of embryo development to postnatal day 30. Tactivin prevented the development of dexamethasone-induced apoptosis in thymocytes of 30-day-old rats, but had no effect on spontaneous apoptosis. Tactivin can be used as a modulator of apoptotic processes.

Immunological Consequences of Apoptotic Cell Phagocytosis

Cells undergo apoptosis in development, tissue homeostasis, and disease and are subsequently cleared by professional and nonprofessional phagocytes. There is now overwhelming evidence that phagocyte function is profoundly altered following apoptotic cell uptake, with consequences for the ensuing innate and adaptive immune response. Pathogens and tumors exploit the changes in macrophage function following apoptotic cell uptake. Here, we will outline the consequences of apoptotic cell phagocytosis and illustrate how apoptotic cells could be used to manipulate the immune response for therapeutic gain.

Glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action.

Prominent blood and tissue eosinophilia is clinically manifested in a number of inflammatory states, particularly in allergic diseases. Corticosteroids are the most effective anti-inflammatory drugs used in the treatment of eosinophilic disorders, including bronchial asthma. Their beneficial effects result, among others, from (i) the suppression of the synthesis and the effects of eosinophil survival factors, (ii) the direct induction of eosinophil apoptosis and (iii) the stimulation of their engulfment by professional phagocytic cells. Failure of steroids to propagate apoptotic signals and to promote eosinophil clearance may explain the corticoresistance observed in a proportion of asthmatic patients. Accordingly, studies on the intracellular mechanisms involved in eosinophil corticosensitivity and resistance may provide a valuable tool for identifying new and selective molecular targets to therapeutically resolve otherwise persistent eosinophilic inflammation. In this review, the intracellular cascade of events involved in corticosteroid-mediated eosinophil apoptotic death is discussed and compared to the signalling pathway governing this process in the established model of dexamethasone-induced thymocyte apoptosis.

Thymosin α 1 is a time and dose-dependent antagonist of dexamethasone-induced apoptosis of murine thymocytes in vitro

It is well established that glucocorticoid hormones induce apoptosis in immature developing thymocytes. Thymocyte apoptotsis can be modulated by growth factors, anti-oxidants and adhesion receptors. We have previously demonstrated that thymosin α 1 (Tα 1) antagonizes dexamethasone-induced apoptosis of CD4 +CD8 + thymocytes. In the present study, we further characterize the dose and time dependence of Tα 1's antagonism of dexamethasone-induced thymocyte apoptosis. Tα 1 is effective at concentrations ranging from 2 to 100 μg/10 6 thymocytes. Tα 1 pre-treatment is necessary to achieve its anti-apoptotic activity. Tα 1 provides temporary protection to thymocytes by slowing dexamethasone's apoptotic activity up to 12 h post dexamethasone treatment. Additionally, Tα 1's activity is not sensitive to cycloheximide treatment, suggesting Tα 1's activity is independent of protein synthesis. Finally, Tα 1 is unable to antagonize apoptosis induced by the reactive oxygen species, H 2O 2, suggesting Tα 1's antagonism of dexamethasone occurs at the early stages of dexamethasone-induced apoptosis, prior to the production of reactive oxygen species. This evidence suggests that Tα 1 may provide a mechanism to transiently extend the life of a thymocyte during thymic selection.

Streptococcal wall component OK432 restores sensitivity of non-obese diabetic (NOD) thymocytes to apoptotic signals.

The streptococcal wall component, OK432, prevents diabetes in NOD mice and BB rats by elimination of effector cells. Based on the knowledge of a link between autoimmunity and resistance of immune cells to elimination by apoptosis, we investigated whether OK432 treatment restored the sensitivity of NOD lymphocytes to apoptotic signals centrally (thymus) or peripherally (spleen) or both and we examined the pathways for the enhanced apoptosis rate. We treated NOD mice with OK432 (0.1 mg/kg i.p. weekly from 21 to 70 days). Apoptosis was measured by TUNEL 16 h after cyclophosphamide (70 mg/kg) and 24 h after dexamethasone (0.2 mg/mouse). Real time quantitative RT-PCR was used to investigate changes in gene expression. Thymocyte apoptosis levels after cyclophosphamide were restored by OK432 treatment to levels observed in C57BL/6 mice: in NOD males apoptosis increased from 8 +/- 1% to 18 +/- 5% (p < 0.05) compared with 20 +/- 4% in C57BL/6 males, and in NOD females from 6+/- 2% to 11 +/- 2% (p < 0.05) compared with 12 +/- 2% in C57BL/6 females. The dexamethasone-induced thymocyte apoptosis rate was equally restored by OK432 treatment (58 +/- 4% vs 41 +/- 3% in control males (p < 0.0005) and 39 +/- 5% vs 26 +/- 3% in control females (p < 0.05)]. No change in apoptosis levels was on the contrary observed in splenocytes after OK432 treatment. By RT-PCR analysis of a panel of apoptosis-related genes in thymocytes we showed a down-regulation of anti-apoptotic Bcl-xL and c-myc by OK432 treatment. Our data suggest that OK432 prevents diabetes in NOD mice by better elimination of effector cells through increased sensitivity to apoptotic signals centrally in the thymus.

Dexamethasone-Induced Thymocyte Apoptosis: Apoptotic Signal Involves the Sequential Activation of Phosphoinositide-Specific Phospholipase C, Acidic Sphingomyelinase, and Caspases

AbstractGlucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein–dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

Dexamethasone-Induced Thymocyte Apoptosis: Apoptotic Signal Involves the Sequential Activation of Phosphoinositide-Specific Phospholipase C, Acidic Sphingomyelinase, and Caspases

Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein–dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

Intracellular alkalinization in dexamethasone-induced thymocyte apoptosis.

Glucocorticoid can induce apoptosis of thymocytes, but its mechanism is not clear yet. In this study, we reported that dexamethasone-induced apoptosis was associated with intracellular alkalinization. Dexamethasone induced a higher percentage of apoptosis in 138 mM than in 50 mM NaCl, total abrogation of apoptosis was noted in NaCl-depleted culture medium. Highest apoptotic rate was observed in medium with pH 7.2, whereas it was partially and completely inhibited at pH 6.5 and pH 6.0, respectively. Intracellular pH was higher in pre-apoptotic thymocytes than non-apoptotic ones. The Na+/H+ antiporter inhibitor of 5-(N,N'-dimethyl)-amiloride inhibited the dexamethasone-induced increase in pHi and apoptosis of thymocytes. Glucocorticoid antagonist RU486 also blocked the dexamethasone-induced effect. Furthermore, the apoptosis and increase in intracellular pH induced by dexamethasone were inhibited by cycloheximide, actinomycin D. It seems that intracellular pH is increased during the development of thymocyte apoptosis and inhibiting its increment would retard the rate of progression to cell death.

Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis.

In this paper we used a multiparametric approach to analyze extensively the events occurring during apoptotic cell death of thymocytes, and furthermore, we asked whether alterations in mitochondrial structure and function are occurring in early stages of apoptosis. A multiparametric quantitative analysis was performed on normal or apoptotic thymocytes emerging from a few-hour culture performed in culture medium or in the presence of dexamethasone. Simultaneous detection of light scattering properties, integrity of plasma membrane (trypan blue exclusion), chromatin condensation (AO/EB staining of entire cells or PI staining of nuclei), and DNA fragmentation (in situ nick-translation in apoptotic cells) allowed a precise analysis of the preapoptotic and apoptotic stages. Moreover a thorough study of mitochondrial transmembrane potential (delta psi m) assessed following in a time course study the uptake by apoptotic cells of the cationic lipophilic dye DiOC6(3) or the J-aggregate-forming cation JC-1, indicates that a drop in delta psi m occurs very early in thymocyte apoptosis, before DNA fragmentation. This is associated with alteration in mitochondrial structure assessed by cytofluorimetric study of NAO uptake in apoptotic cells. Finally these dramatic alterations in mitochondrial structure and function occurring in early stages of apoptosis were confirmed by confocal and electron microscopy analysis.

Extracellular calcium is not required for gliotoxin or dexamethasone-induced DNA fragmentation: A reappraisal of the use of EGTA

The immunomodulating agent gliotoxin and related toxins cause apoptotic cell death in a variety of cell types including macrophages and thymocytes [Waring et al. (1988) J. biol. Chem., 263, 18,493-18,499; Waring et al. (1990) Int. J. Immunopharmac., 12, 445–457]. The mechanism of induction of apoptosis by gliotoxin is not yet known, although it does not require protein synthesis [Waring (1990) J. biol. Chem., 14,476-14,480], unlike dexamethasone-induced apoptosis in thymocytes. Because of the reported requirement for extracellular calcium in apoptosis induced by dexamethasone, we studied the effects of extracellular calcium on gliotoxin-induced apoptosis in macrophages. Initial experiments using calcium-depleted media showed no inhibition of apoptotic DNA fragmentation by gliotoxin. By measuring residual 45Ca2+ remaining in cells pulsed with labelled calcium over the time period required for DNA fragmentation, we could demonstrate some uptake of extracellular calcium into treated cells as assessed by residual, slowly exchanging calcium. When cells were treated with the calcium chelator EGTA at 0.5-2 mM, calcium uptake was abolished but DNA fragmentation was unaffected. EGTA at higher concentrations, up to 8 mM, did inhibit DNA fragmentation without any additional inhibition of calcium uptake. Similar results were found for dexamethasone-treated thymocytes. Thymocytes treated with 8 mM EGTA, however, were not rescued from apoptosis but died by necrosis. These results indicate that extracellular calcium is not essential for apoptosis induced by these agents and that the use of high concentrations of EGTA to establish a requirement for extracellular calcium in apoptosis should be treated with caution.

Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.

Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation.

Apoptosis is a major form of cell death, characterized morphologically by chromatin condensation and biochemically by endonuclease cleavage of DNA into oligonucleosomal fragments. Recently, we reported that zinc arrested dexamethasone-induced apoptosis in thymocytes at an early stage, as characterized morphologically by condensation of heterochromatin into clumps abutting the nuclear membrane. In this study, we show that zinc completely inhibits endonuclease cleavage of DNA into oligonucleosomal fragments but does not prevent the cleavage of DNA into high molecular weight fragments. These results indicate that the formation of these high molecular weight fragments, which correlates with the very early morphological features of apoptosis, is a critical event in glucocorticoid-induced apoptosis. The formation of these high molecular weight fragments, despite the inhibition by zinc of the endonuclease cleavage of DNA, suggests that key enzyme(s), other than the Ca2+/Mg(2+)-dependent endonuclease, are involved at the earliest stages of induction of apoptosis.

Pidotimod stimulates natural killer cell activity and inhibits thymocyte cell death.

Experiments were performed to analyze the possible effect of the immunomodulating agent Pidotimod (3-L-pyroglutamyl-L-thiazolidine-4-carboxylic acid) on mouse Natural Killer (NK) cell activity and glucocorticoid hormone(GCH)-induced thymocyte apoptosis. The results indicate that in vivo treatment with Pidotimod (200 mg/Kg ip for 5 days) causes a significant increase in NK activity and in vitro treatment produces a significant reduction of dexamethasone-induced thymocyte apoptosis. This inhibition appears to be dose-dependent and is also evident against TPA or Ca(++)ionophore-induced apoptosis.