Cycloastragenol Exerts an Antiapoptotic Effect on Human Lymphocytes under UV-Irradiation

CD14 is a 56 Kd glycoprotein typically present on myeloid cells and originally implicated in innate immunity and pattern recognition. The possible involvement of its membrane bound form with the clearance of apoptotic cells and its recently demonstrated expression by non-myeloid cells, make this fascinating molecule a target for more research and possible therapeutic use. The objective of this study was to assess the possible anti apoptotic effect of the soluble form of CD14, on human lymphocytes and to delineate the fragment, from within the protein that may be mediating that activity. Human peripheral blood lymphocytes were cultured in vitro and induced for apoptosis with gliotoxin, with or without the addition of rhCD14 or CD14 peptide. As controls, we used lymphocyte cell lines such as Jurkat, NK-YTS and also normal peripheral blood neutrophils. We demonstrate that human soluble CD14 is capable of protecting human lymphocytes from gliotoxin- induced apoptosis in vitro. It is not capable of protecting gliotoxin-treated apoptotic Jurkat or NK-YTS cells. Moreover, we reveal that a fragment of the protein, a peptide that is recognized by the MO2 monoclonal anti-CD14 antibody, retains this protective effect, although with a lower efficiency. We have previously shown that the MO2 epitope was found naturally inside human lymphocytes. Here, we demonstrate that the MO2 peptide can penetrate lymphocytes in human peripheral blood mononuclear cells incubated in vitro. The peptide does not penetrate Jurkat or NK-YTS cells and it does not protect them from apoptosis. In addition, peripheral blood neutrophils, in which the peptide penetration was much better than in lymphocytes, could not be protected by this peptide from their spontaneous apoptosis in vitro.Our data thus suggest that circulating CD14 may play an important role in the prevention of apoptosis of lymphocytes and that a specific region from within the molecule is involved in this activity.

Changes in the level of some markers of apoptosis (lipid asymmetry of plasma membranes, production of reactive oxygen species, nitric oxide, concentration of ionized calcium) of human blood lymphocytes modified by exposure to hydrogen peroxide (10 mol/l) and UV light (254 nm, 1510 J/m) in the presence of cycloastragenol (10–10 mol/l) were studied. The intensity of apoptotic lymphocyte death processes was found to decrease after UV-light exposure in the presence of cycloastragenol (10 mol/l). The cytoprotective effect of cycloastragenol on lymphocytes was found to be due to a decrease in the level of intracellular reactive oxygen species and calcium ions, an increase in the activity of catalase and glutathione reductase and nitric oxide production. Possible mechanisms of cycloastragenol action as a regulator of apoptotic death of lymphocytes induced by UV radiation and hydrogen peroxide exposure are discussed.

Protective effect of Zingerone, a dietary compound against radiation induced genetic damage and apoptosis in human lymphocytes

Anti-Apoptotic Effects of Platelet Factor 4 on Human T-Lymphocytes

Ginkgo biloba (EGb 761) is a well-defined plant extract that directly scavenges hydroxyl radicals. It is a potent antioxidant that inhibits apoptosis in cultured cells and is effective in treating mild-to-moderate dementia in Alzheimer patients. Apoptosis is an active process of cell destruction and it plays an important role in pathological processes. The aim of this study is to investigate the anti-apoptotic effect of EGb 761 in gossypol-treated human lymphocytes. Pretreatment of lymphocytes with 10 microg/ml EGb 761 for 30 min or 1h decreased the percentage of apoptosis to 17.5% and 20%, respectively. EGb 761 treatment (25-150 microg/ml) decreased the level of apoptosis to a plateau between 8 and 10% of the control values. We conclude that EGb 761 reduces gossypol-induced apoptosis in human lymphocytes.

Regulating apoptosis of lymphocytes is an effective strategy for treatment of lymphocyte-mediated diseases. Recently it has been demonstrated that augmenter of liver regeneration (ALR), an enigmatic protein presented ubiquitously in multiple forms among eukaryotes, possesses potent anti-apoptotic activity and supports proliferation of a variety of cells. However, its action on lymphocytes and the underlying mechanism are not completely understood. In this study, we analyzed the effects of recombinant human ALR (rhALR) on apoptosis of human lymphocytes activated with concanavalin A (ConA). Our results showed that rhALR inhibited apoptosis of ConA-activated lymphocytes and revealed reductions in the percentage of apoptotic cells, caspase-3 activation and PARP cleavage in cells treated with rhALR. Furthermore, the BAX/BCL-2 and cytosol/mitochondria cytochrome c ratios were decreased in the intrinsic death pathway and the activation of caspase-8 was also decreased in the extrinsic death pathway in activated lymphocytes treated with rhALR. In addition, rhALR significantly reduced the quantity of interleukin-2. These results demonstrated that rhALR has anti-apoptotic effects on activated lymphocytes through the activation of several apoptosis-related signaling pathways, and shed some light on the effects of rhALR on modulation immune reactions.

Antiapoptotic effect of human T-cell leukemia virus type 1 tax protein correlates with its creb transcriptional activity

Platelet factor 4 (PF4) pertains to a family of CXC chemokines released by activated platelets. PF4 has a broad spectrum of effects on different cell types, including modulation of the immune response. In this study, we explore effects of PF4 on the morphological and biochemical signs of apoptosis in human T-lymphocytes in vitro. T-lymphocytes were isolated from peripheral blood of healthy donors using negative immunomagnetic separation and cultured in the complete RPMI 1640 medium for 24 hours in the absence and presence of PF4 added at a final concentration of 2 /ug/ml or 100 ug/ml. After 2, 4, 6, 12 and 24 hours of incubation the cells were studied with transmission electron microscopy and Western blot analysis with respect to potential apoptotic changes. The electron microscopy of control T-lymphocytes showed that the vast majority of the cells had a morphology characteristic of apoptosis at different stages. Adding PF4 at a concentration of 2 ug/ml reduced the number of cells at the late stages of apoptosis, while maintaining the signs of the early apoptosis in most of the T-lymphocytes. In the presence of 100 ug/ml PF4 nearly all of the cells kept a typical morphology of normal T-cells throughout the time of cultivation. The morphological apoptotic changes correlated well with expression of caspase-3, which was clearly detected in the control cells and cells treated with 2 ug/ ml PF4, but was almost abolished in the cells treated with 100 ug/ml PF4. Our results provide direct evidence for the dose-dependent anti-apoptotic effects of PF4 on T-cells, suggesting that PF4 sustains an immune response by extending T-lymphocyte survival.

In the present study we demonstrate that flupirtine, an already clinically used, centrally acting, non-opiate analgesic agent, protects rat cortical neurons against HIV-gp120 induced apoptotic cell death. The drug was active at concentrations between 1 and 10 microg/ml. Furthermore we show inhibition of in vitro induced apoptosis in human blood mononuclear cells, using flupirtine. Induced apoptosis in peripheral blood mononuclear cells from healthy individuals and HIV-1 infected patients was reduced to approximately 50% after in vitro preincubation with flupirtine at concentrations between 0.1 and 10 microg/ml. The anti-apoptotic effect of flupirtine was restricted to CD3+ lymphocytes and in particular to CD4+ cells. Flupirtine does not affect uninduced apoptosis in human lymphocytes in vitro. The selective potential of flupirtine to reduce apoptosis without influencing uninduced apoptosis may qualify this compound as a potential drug in the therapy of HIV-1 infected patients.

Post-IL2 receptor blockade increases fas ligand expression and apoptosis in allospecifically-activated human helper T cells

Homeostasis within the adaptive immune system is a highly controlled process to avoid lymphoproliferation and immune-pathology. Controlled T- and B- cell death in the presence or absence of infection represents an important means to ensure adaptive immune cell homeostasis. Nur77, a nuclear orphan receptor of the NR4A subfamily, is rapidly induced following activation of T-cells and has been demonstrated to modulate apoptosis via diverse cellular mechanisms. Although no endogenous Nur77 ligand could be identified to date, pharmacological targeting of Nur77 with hydrophobic small molecule compounds such as the fungus-derived Cytosporone B and its derivate THPN (1-(3,4,5-trihydroxyphenyl)nonan-1-one) have been reported to induce apoptosis in various cancer types. Here we report experimental evidence, that the Cytosporone B derivate TMPA (ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl) phenyl] acetate) in human and murine primary T-cell blasts dose-dependently impairs restimulation induced cell death (RICD) in a Nur77-independent manner. In addition, TMPA downregulates Nur77-dependently the intracellular adapter protein SAP by reducing mRNA expression of its gene SH2D1A. SAP is essential for immune control of EBV in humans and is critically involved in T-cell-dependent antibody- and auto-antibody formation. To our knowledge, these data show for the first time, that T-cell intrinsic small molecule compound-mediated targeting of Nur77 induces a Nur77-independent antiapoptotic effect and a Nur77-dependent SAP downregulation. This evidence creates the basis to further investigate the therapeutic potential of targeting Nur77 with small molecule compounds, such as TMPA, in immune-control of tumors. Such compounds may induce Nur77-dependent apoptosis in cancer cells and at the same time reduce RICD of cancer-specific T-cells. Furthermore, Nur77-specific small molecule compounds may improve T-cell-dependent vaccinations (e.g. against HIV) or may limit in vivo apoptosis of adoptively transferred tumor-specific T-cells. The modulation of SAP expression by Nur77 binding small molecule compounds may be used to limit autoantibody formation in human autoimmune diseases.

Immunomodulatory and anti-apoptotic effects of Viscum album lipophilic extract and oleanolic acid on human peripheral blood lymphocytes and monocytes in vitro

Leptin (Ob) is a non-glycosylated peptide hormone that regulates energy homeostasis centrally, but also has systemic effects including the regulation of the immune function. We have reported previously that leptin activates human peripheral blood lymphocytes co-stimulated with phytohaemagglutinin (PHA) (4 microg/ml), which prevented the employment of pharmacological inhibitors of signalling pathways. In the present study, we used Jurkat T cells that responded to leptin with minimal PHA co-stimulation (0.25 microg/ml). The long isoform of leptin receptor is expressed on Jurkat T cells and upon leptin stimulation, the expression of early activation marker CD69 increases in a dose-dependent manner (0.1-10 nM). We have also found that leptin activates receptor-associated kinases of the Janus family-signal transucers and activators of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) signalling pathways. Moreover, we sought to study the possible effect of leptin on cell survival and apoptosis of Jurkat T cells by culture in serum-free conditions. We have assayed the early phases of apoptosis by flow cytometric detection of fluorescein isothiocyanate (FITC)-labelled annexin V simultaneously with dye exclusion of propidium iodide (PI). As well, we have assayed the activation level of caspase-3 by inmunoblot with a specific antibody that recognizes active caspase-3. We have found that leptin inhibits the apoptotic process dose-dependently. By using pharmacological inhibitors, we have found that the stimulatory and anti-apoptotic effects of leptin in Jurkat T cells are dependent on MAPK activation, rather than the PI3K pathway, providing new data regarding the mechanism of action of leptin in T cells, which may be useful to understand more clearly the association between nutritional status and the immune function.

RNA synthesis inhibitors and protein synthesis inhibitors are useful for investigating whether biological events with unknown mechanisms require transcription or translation; however, the dependence of RNA synthesis has been difficult to verify because many RNA synthesis inhibitors cause adverse events that trigger a p53 response. In this study, we screened a library containing 9600 core compounds and obtained STK160830 that shows anti-apoptotic effects in irradiated wild-type-p53-bearing human T-cell leukemia MOLT-4 cells and murine thymocytes. In many of the p53-impaired cells and p53-knockdown cells tested, STK160830 did not show a remarkable anti-apoptotic effect, suggesting that the anti-apoptotic activity is p53-dependent. In the expression analysis of p53, p53-target gene products, and reference proteins by immunoblotting, STK160830 down-regulated the expression of many of the proteins examined, and the downregulation correlated strongly with its inhibitory effect on cell death. mRNA expression analyses by qPCR and nascent RNA capture kit revealed that STK160830 showed a decreased mRNA expression, which was similar to that induced by the RNA synthesis inhibitor actinomycin D but differed to some extent. Furthermore, unlike other RNA synthesis inhibitors such as actinomycin D, p53 accumulation by STK160830 alone was negligible, and a DNA melting-curve analysis showed very weak DNA-intercalating activity, indicating that STK160830 is a useful inhibitor for RNA synthesis without triggering p53-mediated damage responses.

Melatonin is considered a promising antitumor agent, promoting apoptosis in tumor cells and contrasting it in normal cells. The basis for this selectivity is presumed to be the ability of melatonin to stimulate reactive oxygen species (ROS) production in tumor cells. Here we investigate the effect of melatonin on three types of human lymphocytes: normal blood lymphocytes, BL41 Burkitt lymphoma, and the cognate Epstein-Barr virus (EBV)-converted E2r. We found that melatonin promotes ROS production in all these cells. Melatonin protects BL41 from apoptosis in the same manner as normal lymphocytes, whereas E2r are unaffected. These results show that ROS production is not limited to tumor lymphocytes nor it is involved in apoptosis promotion; that melatonin does not promote apoptosis in tumor lymphocytes, but EBV inhibits melatonin anti-apoptotic effects; and that the anti-apoptotic effect of melatonin does not depend on the well-known chemical antioxidant properties of melatonin.