Glutamine protects activated human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels

Glutamine is the most abundant amino acid in the body. A decrease of plasma glutamine concentrations is found in catabolic stress and is related to susceptibility to infections. Glutamine is known to modulate lymphocyte activation; however, little is known about glutamine modulation of cell death of activated human T cells. Using Jurkat T cells, we investigated glutamine modulation of T-cell apoptosis activated by PMA plus ionomycin. We found that glutamine at various concentrations significantly enhanced IL-2 production, cell proliferation, and cell viability of Jurkat T cells. Glutamine also decreased the number of apoptotic cells stimulated with PMA plus ionomycin as demonstrated by flow cytometry. Meanwhile, glutamine down-regulated CD95 and CD95L expression, but up-regulated CD45RO and Bcl-2 expression in activated T cells. Further investigation of CD95-mediated caspase activities revealed that supplementation of glutamine significantly decreased caspase-3 and caspase-8 activities in activated T cells. Since oxidative stress is closely associated with induction of lymphocyte apoptosis, we found that glutamine significantly increased glutathione (GSH), but decreased reactive oxygen species levels in activated T cells. Blockade of intracellular GSH formation enhanced, but exogenous GSH supplementation decreased, activated T-cell apoptosis. Studying normal peripheral lymphoproliferation, we also found that the presence of glutamine increased lymphoproliferation as well as Bcl-2 and CD95 expression; but decreased CD95L and activation-induced T-cell death. Taken together, glutamine appeared to augment lymphoproliferation but suppressed activation-induced T-cell death in both Jurkat T cells and human peripheral T lymphocytes.     

Morphine promotes apoptosis in Jurkat cells.

Patients with intravenous heroin addiction are prone to recurrent infections and at times these infections are fatal. We evaluated the effect of morphine on the apoptosis of Jurkat cells and freshly isolated human T lymphocytes. Morphine promoted apoptosis of both the Jurkat cells and the freshly isolated T lymphocytes in a dose-dependent manner. DAGO, a specific mu receptor agonist, also promoted Jurkat cell apoptosis. DNA isolated from morphine-treated Jurkat cells and T lymphocytes also showed integer multiples of 200 base pairs. Superoxide dismutase (SOD) enhanced lymphocyte apoptosis; whereas catalase attenuated the morphine-induced apoptosis of Jurkat cells as well as of T lymphocytes. Morphine-treated Jurkat cells also showed a decreased expression of bcl-2 and an enhanced expression of bax. In addition, morphine-treated Jurkat cells showed activation of caspase-3. These results indicate that morphine-induced T lymphocyte apoptosis may be mediated through the generation of reactive oxygen species. The change in ratio of bax and bcl-2 seems to tilt the balance toward apoptosis, leading to the activation of caspase-3. This study provides further support for the hypothesis that morphine may be directly compromising immune function by enhancing apoptosis of T lymphocytes in patients with heroin addiction.     

Ethanol-induced neutrophil apoptosis is mediated through nitric oxide

Clinical reports indicate that acute ethanol intoxication in chronic ethanol abusers is associated with neutropenia. We hypothesize that ethanol accelerates the apoptosis of neutrophils thus decreasing the peripheral blood count of neutrophils. We studied the effect of ethanol on neutrophil apoptosis in vivo as well as in vitro. Human neutrophils harvested from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 0.5+/-0.25 vs. after, 26.1+/-2.6% apoptotic neutrophils/field). Peritoneal neutrophils isolated from ethanol-treated rats also showed increased (P < 0.0001) apoptosis when compared with neutrophils isolated from control rats (control, 0.8+/-0.2% vs. ethanol, 11.8+/-0.7% apoptotic neutrophils/field). In in vitro studies, ethanol in concentrations of 50 mM and higher accelerated the apoptosis of human and rat neutrophils. This effect of ethanol on human neutrophils was time dependent. DNA isolated from ethanol-treated human neutrophils displayed integer multiples of 180 base pairs (ladder pattern), further confirming the effect of ethanol on neutrophil apoptosis. N(G)-monomethyl-L-arginine monoacetate and N(G)-nitro-L-arginine methyl ester, inhibitors of nitric oxide (NO) synthase, attenuated the ethanol-induced neutrophil apoptosis. Sodium nitroprusside, a NO donor, also promoted neutrophil apoptosis. Moreover, ethanol enhanced neutrophil expression of inducible NO synthase. In addition, ethanol stimulated neutrophil NO generation. These results suggest that ethanol accelerates neutrophil apoptosis. This effect of ethanol on neutrophil apoptosis seems to be mediated through the generation of NO.     

Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.      

Biochemical and molecular basis of thimerosal-induced apoptosis in T cells: a major role of mitochondrial pathway

The major source of thimerosal (ethyl mercury thiosalicylate) exposure is childhood vaccines. It is believed that the children are exposed to significant accumulative dosage of thimerosal during the first 2 years of life via immunization. Because of health-related concerns for exposure to mercury, we examined the effects of thimerosal on the biochemical and molecular steps of mitochondrial pathway of apoptosis in Jurkat T cells. Thimerosal and not thiosalcylic acid (non-mercury component of thimerosal), in a concentration-dependent manner, induced apoptosis in T cells as determined by TUNEL and propidium iodide assays, suggesting a role of mercury in T cell apoptosis. Apoptosis was associated with depolarization of mitochondrial membrane, release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria, and activation of caspase-9 and caspase-3, but not of caspase-8. In addition, thimerosal in a concentration-dependent manner inhibited the expression of XIAP, cIAP-1 but did not influence cIAP-2 expression. Furthermore, thimerosal enhanced intracellular reactive oxygen species and reduced intracellular glutathione (GSH). Finally, exogenous glutathione protected T cells from thimerosal-induced apoptosis by upregulation of XIAP and cIAP1 and by inhibiting activation of both caspase-9 and caspase-3. These data suggest that thimerosal induces apoptosis in T cells via mitochondrial pathway by inducing oxidative stress and depletion of GSH.     

Glucose enhances mesangial cell apoptosis

Mesangial cell apoptosis occurs in experimental diabetic nephropathy, and this correlates with worsening albuminuria. This study examines the mechanism by which glucose modulates mesangial cell apoptosis. Apoptosis was induced in mesangial cells by serum deprivation in the presence of 5 or 25 mM D-glucose, and examined by expression of Annexin-V and disruption of mitochondrial transmembrane potential. Involvement of Bax, Bcl-2 and NF-kappaB were examined by RT-PCR and EMSA. Involvement of TGF-beta1 was sought by determining the effect of recombinant TGF-beta1on apoptosis and the mediators of the apoptotic pathway (Bcl2/Bax and NF-kappaB). Culture of cells in the presence of 25 mM D-glucose (i) enhanced apoptosis stimulated by serum depletion, (ii) enhanced activation of caspase-3, (iii) inhibited NF-kappaB activation, and (iv) decreased Bcl-2:Bax ratio. Inhibition of NF-kappaB using SN50, also increased mesangial cell apoptosis, and decreased Bcl-2:Bax ratio. Addition of TGF-beta1 to mesangial cells mimicked the effect of high glucose reducing NF-kappaB expression and Bcl-2:Bax ratio. Furthermore glucose-mediated enhanced apoptosis was inhibited by the addition of a blocking antibody to TGF-beta1. Exposure of mesangial cells to 25 mM D-glucose stimulated the generation of both total and active TGF-beta1 in the cell culture supernatant, this increase was only significant after 48-72 h, that is at a time point later than enhanced apoptosis. Addition of 25 mM D-glucose, however, increased sensitivity of mesangial cells to TGF-beta1 as assessed by luciferase activity of a Smad sensitive reporter construct. The data suggest that elevated glucose concentration enhanced the pathway leading to apoptosis following serum deprivation. Furthermore, it is likely that this is dependent on glucose-mediated enhanced sensitivity to endogenous TGF-beta1 rather than glucose stimulated de novo TGF-beta1 synthesis.     

Caspase-2 and caspase-7 are involved in cytolethal distending toxin-induced apoptosis in Jurkat and MOLT-4 T-cell lines

Cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is a G2/M cell-cycle-specific growth-inhibitory toxin that leads to target cell distension followed by cell death. To determine the mechanisms by which A. actinomycetemcomitans CDT acts as an immunosuppressive factor, we examined the effects of highly purified CDT holotoxin on human T lymphocytes. Purified CDT was cytolethal toward normal peripheral T lymphocytes that were activated by in vitro stimulation with phytohemagglutinin. In addition, purified CDT showed cytolethal activity against Jurkat and MOLT-4 cells, which are known to be sensitive and resistant, respectively, to Fas-mediated apoptosis. Death in these cell lines was accompanied by the biochemical features of apoptosis, including membrane conformational changes, intranucleosomal DNA cleavage, and an increase in caspase activity in the cells. Pretreatment of Jurkat cells with the general caspase inhibitor z-VAD-fmk mostly suppressed CDT-induced apoptosis. Furthermore, specific inhibitors of caspase-2 and -7 showed significant inhibitory effects on CDT-induced apoptosis in Jurkat cells, and these inhibitory effects were fully associated with reduced activity of caspase-2 or -7 in the CDT-treated Jurkat cells. These results strongly suggest that CDT possesses the ability to induce human T-cell apoptosis through activation of caspase-2 and -7.     

Spontaneous and oxidative stress-induced programmed cell death in lymphocytes from patients with ataxia telangiectasia (AT)

T cell lymphopenia in the peripheral blood lymphocytes (PBL) of patients with AT is mainly caused by a decrease of naive CD45RA+/CD4+ cells followed by a predominance of memory CD45RO+ lymphocytes. To relate these findings to the regulation of programmed cell death, we investigated the activation state and apoptotic level of PBL in 12 patients and healthy controls by flow cytometry. In accordance with previous investigations, the number of naive CD4+/CD45RA+ cells was significantly decreased in patients compared with healthy controls. This disturbed balance of CD45RA and CD45RO was also reflected in higher amounts of activated HLA-DR and CD95 expressing cells, with a concomitant decrease of Bcl-2 protected lymphocytes in the T cell population. With regard to its role in preventing oxidative-induced cell death, we analysed Bcl-2 expression and apoptosis in the presence of oxidative stress. In culture, cells of patients are more susceptible to spontaneous programmed cell death. However, in our stress-inducing system (hypoxanthine/xanthine oxidase system) the number of cells undergoing apoptosis was lower in patients' cell populations compared with controls. In addition, preliminary results suggest that Bcl-2 expression and level of spontaneous apoptosis in patients can be modified by IL-2 and interferon-gamma.     

Synthetic liver X receptor agonist T0901317 attenuates high glucose-induced oxidative stress,mitochondrial damage and apoptosis in cardiomyocytes

The aim of the present study was to investigate the protective effects of T0901317 (T0), a potent agonist of liver X receptors (LXRs), on high glucose-induced oxidative stress and apoptosis in H9c2 cardiac cells. Exposure of H9c2 cells to high glucose alone, not only caused a significant increase in apoptosis and reactive oxygen species (ROS) generation, but also led to a decrease in mitochondrial membrane potential (ΔΨm), release of cytochrome c, decrease in Bcl-2, increase in Bax expression and the activation of caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP) and nuclear factor (NF)-κB. However, pretreatment with T0 effectively decreased apoptosis, reduced the levels of ROS, abrogated ΔΨm, inhibited cytochrome c release and NF-κB activation, increased Bcl-2 and decreased Bax expression. In conclusion, our data suggest that T0 exerts protective effects against high glucose-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS production, mitochondrial death and NF-κB activation.     

小檗碱增强阿霉素诱导的膀胱癌T24细胞凋亡

 目的:研究小檗碱对阿霉素诱导的膀胱癌T24细胞凋亡的影响及机制。方法:将膀胱癌T24细胞分为对照组、阿霉素组、阿霉素+小檗碱组和小檗碱组。采用CCK-8试剂盒测定膀胱癌T24细胞的增殖抑制率。采用Hoechst 33258染色剂检测细胞凋亡,同时测定caspase-3和caspase-9活性以及Bcl-2、Bax蛋白的表达。结果:小檗碱呈剂量和时间依赖性地促进阿霉素诱导的T24细胞凋亡。与阿霉素组比较,小檗碱+阿霉素组caspase-3、caspase-9活性和Bax蛋白的表达水平明显增加,而Bcl-2蛋白表达水平降低。结论:小檗碱可进一步增强阿霉素对T24细胞增殖的抑制作用,其机制与小檗碱增强阿霉素诱导的T24细胞凋亡有关。     

川楝素与γδ T细胞协同抗结直肠癌的作用及机制研究

目的:探讨γδT细胞对结直肠癌细胞的杀伤活性,并研究川楝素对γδT细胞的协同效应。方法:体外培养人γδT细胞并用流式细胞术进行鉴定。乳酸脱氢酶(LDH)释放实验检测γδT细胞和川楝素对人结直肠癌SW480细胞的杀伤活性。Western blot实验检测川楝素对SW480细胞中Bcl-2、Bcl-x L和MCL-1表达水平的影响。ELISA实验检测川楝素是否影响γδT细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)和Fas配体(Fas L)的分泌。流式细胞术检测川楝素和γδT细胞处理后SW480细胞的线粒体膜电位和凋亡情况。Western blot实验检测川楝素和γδT细胞处理后SW480细胞caspase-9和caspase-3的活化。结果:体外培养的γδT细胞高表达CD3和γδT细胞受体(TCR)。川楝素处理能显著增强γδT细胞对SW480的杀伤活性。川楝素不影响γδT细胞TRAIL和Fas L的表达。川楝素不影响SW480细胞中Bcl-2和Bcl-x L的蛋白水平但显著抑制MCL-1的表达。转染MCL-1质粒能显著抑制川楝素对γδT细胞的协同效应。川楝素通过抑制MCL-1的表达促进γδT细胞对SW480细胞线粒体膜电位的损伤和凋亡的诱导。川楝素通过抑制SW480细胞MCL-1的表达促进γδT细胞对SW480细胞caspase-9和caspase-3的活化。结论:川楝素通过抑制MCL-1的表达发挥对γδT细胞的协同抗结直肠癌作用。     

Synergistic action of protein kinase C theta and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells

Deletion of activated peripheral T cell clones by apoptosis requires the regulated expression of Fas ligand (FasL) and sensitization of these cells to CD95-mediated signaling. To investigate the signaling pathways responsible for FasL expression in T cells, we tested-besides subfamily-selective protein kinase C (PKC) inhibitors - the effect of constitutively active mutants of representatives of all PKC subfamilies, i.e. PKCalpha,epsilon,theta,iota, on FasL luciferase promoter reporter constructs. In synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCtheta, but not PKCalpha,epsilon,iota, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCtheta AE-estrogen receptor fusion mutant led to a CaN-dependent and rapid FasL reporter activity detected as early as 4 h after addition of 4-hydroxytamoxifen, incidating a direct effect of PKCtheta action on FasL expression. Consistently, in Jurkat T cells, expression of PKCtheta AE / CaN significantly enhanced FasL protein expression and apoptosis in a CD95-dependent manner since cell death was not observed in T cells co-expressing the caspase-8 inhibitor crmA. Taken together, our results support the notion that PKCtheta and CaN are sufficient to regulate apoptosis through FasL expression.     

Ethanol-Induced Apoptosis in Mouse Liver : Fas- and Cytochrome c-Mediated Caspase-3 Activation Pathway

Hepatic apoptosis has been shown to occur in both experimental and clinical alcoholic liver disease, but the signaling pathway remains unknown. This study was undertaken to examine specifically the involvement of the upstream signals, Fas and cytochrome c, in alcohol-induced caspase-3 activation and apoptosis in the liver. Male FVB mice were administrated intragastrically a single dose of alcohol at 6 g/kg, which has been shown to represent binge drinking in humans. Hepatic apoptosis was detected by a terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Active form of caspase-3 was identified by immunoperoxidase staining and confirmed by immunogold labeling and was found to be in the cytosol and nucleus. Enzymic assay further confirmed caspase-3 activation and nucleus localization. Systemic administration of caspase-3 inhibitor, Ac-DEVD-FMK, inhibited caspase-3 activity and abrogated apoptosis. Elevation of cytosolic cytochrome c was found by immunoperoxidase staining, immunogold labeling, and Western blot. Increased Fas ligand expression was detected by immunoperoxidase staining. Intravenous administration of a neutralizing Fas ligand monoclonal antibody resulted in suppression of caspase-3 activation and attenuation of apoptosis, but did not inhibit mitochondrial cytochrome c release. The results thus demonstrate that Fas/Fas ligand system-mediated caspase-3 activation plays a central role in the ethanol-induced hepatic apoptosis.     

Aberrant integrin activation induces p38 MAPK phosphorylation resulting in suppressed Fas-mediated apoptosis in T cells: Implications for rheumatoid arthritis

Delayed Fas-mediated apoptosis in T cells is associated with inflammatory diseases including rheumatoid arthritis (RA). CD3⁺ T cells in RA synovia expressed high amounts of phospho-p38 MAPK. Exposure to RA synovial fluid or soluble collagen, a degradation product of extracellular matrix abundant in RA synovium, induced the phosphorylation of p38 MAPK in Jurkat T cells accompanied by resistance against Fas-mediated apoptosis. Blocking β1 integrin by antibody diminished this effect. In addition, ectopic expression of auto-activated β1 integrin variant in T cells profoundly induced the phosphorylation of p38 MAPK. Suppression of p38 MAPK sensitized T cells to Fas-mediated apoptosis and increased caspase-8 and caspase-3 cleavage. A physical interaction of p38 MAPK and caspase-8 was demonstrated by using confocal microscopic imaging and co-immunoprecipitation assay. RA synovial fluid markedly increased the formation of phospho-p38 MAPK/caspase-8 complex in Jurkat T cells. In conclusion, abnormal activation of p38 MAPK to prevent Fas-mediated apoptosis may represent a common survival mechanism of RA synovial T cells contributing to the persistent inflammation of affected synovium.     

Angiotensin-converting enzyme inhibitor captopril prevents activation-induced apoptosis by interfering with T cell activation signals

Captopril is an orally active inhibitor of angiotensin-converting enzyme (ACE) which is widely used as an anti-hypertensive agent. In addition to its ability to reduce blood pressure, captopril has a number of other biological activities. Recently the drug was shown to inhibit Fas-induced apoptosis in human activated peripheral T cells and human lung epithelial cells. In this study, we investigated whether captopril blocks activation-induced apoptosis in murine T cell hybridomas, and found that captopril inhibited IL-2 synthesis and apoptotic cell death upon activation with anti-CD3 antibody. In addition, captopril inhibited an inducible caspase-3-like activity during activation-induced apoptosis. On the other hand, captopril did not interfere with Fas signalling, since anti-Fas antibody-induced apoptosis in Fas+ Jurkat cells was unaffected by the drug. Furthermore, we examined whether captopril blocks activation-induced apoptosis by interfering with expression of Fas, Fas ligand (FasL), or both on T cell hybridomas. FasL expression on activated T cells was significantly inhibited by captopril, whereas up-expression of Fas was partially inhibited, as assessed by cell surface staining. Taking all data together, we conclude that captopril prevents activation-induced apoptosis in T cell hybridomas by interfering with T cell activation signals. Captopril has been reported to induce systemic lupus erythematosus syndrome, and our findings may be useful for elucidating the mechanism of captopril-induced autoimmunity.     

Mechanisms Underlying Production And Externalization of Oxidized Phosphatidylserine in Apoptosis: Involvement of Mitochondria

The present study was performed by using selective inhibitors of caspase-8 and caspase-3 functioning upstream and downstream from mitochondria, respectively to determine whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, release of cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced mitochondrial cyt c release, the amount of PSox not only within but also on the surface of Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on the surface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation and apoptosis. Thus, these results strongly suggest that mitochondria play an important role in production of PSox and subsequent its externalization during apoptosis.     

左旋门冬酰胺酶与盐霉素联合作用对急性T淋巴细胞白血病Jurkat细胞株增殖凋亡的影响

 目的：研究左旋门冬酰胺酶（L-asparaginase,L-ASP）与盐霉素（salinomycin,Sal）联合作用对急性T淋巴细胞白血病Jurkat细胞株增殖和凋亡的影响及其机制。方法：CCK-8试剂盒检测细胞增殖情况;Western blotting方法检测凋亡相关蛋白Bcl-2、caspase-3、caspase-8、caspase-9及细胞色素C表达的变化;流式细胞术分析各实验组细胞凋亡率之间的差别。结果：不同浓度的L-ASP和Sal单独处理Jurkat细胞株后均显示出明显的增殖抑制作用,L-ASP的IC50为812 IU/L,Sal 的IC50为0.75 μmol/L。而两药联合使用的增殖抑制作用更显著（P<0.05）;合用指数计算公式结果显示两药为协同作用。Western blotting 显示联合用药组与L-Asp、Sal单独处理组相比,Bcl-2蛋白表达明显减少,caspase-3、caspase-8、caspase-9和胞浆细胞色素C水平明显增高;干预48 h后行流式细胞术检测结果显示L-ASP（25 IU/L）单独处理组和Sal（0.5μmol/L）单独处理组细胞凋亡率分别为（7.11±0.23）%和（25.43±047）%,与对照组（6.67±0.13）%比较差别有统计学意义（P＜0.05),联合用药组细胞凋亡率（39.12±1.97）%分别与L-ASP、Sal单独处理组比较,差别有统计学意义（P＜0.05)。结论：左旋门冬氨酸酶与盐霉素联合作用于Jurkat细胞具有协同抑制增殖和诱导凋亡的作用。     

Butyric acid-induced T-cell apoptosis is mediated by caspase-8 and -9 activation in a Fas-independent manner

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.     

Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.     

Lipopolysaccharide (LPS) Induces the Apoptosis and Inhibits Osteoblast Differentiation Through JNK Pathway in MC3T3-E1 Cells

Bone degradation is a serious complication of chronic inflammatory diseases such as septic arthritis, osteomyelitis, and infected orthopedic implant failure. Up to date, effective therapeutic treatments for bacteria-caused bone destruction are limited. In our previous study, we found that LPS promoted osteoclast differentiation and activity through activation of mitogen-activated protein kinases (MAPKs) pathway such as c-Jun N-terminal kinases (JNK) and extracellular signal regulated kinase (ERK1/2). The current study was to evaluate the mechanism of LPS on the apoptosis and osteoblast differentiation in MC3T3-E1 cells. MC3T3-E1 osteoblasts were non-treated, treated with LPS. After treatment, the cell viability, the activity of alkaline phosphatase (ALP) and caspase-3 were measured. The expressions of osteoblast-specific genes and Bax, Bcl-2, and caspase-3 were determined by real-time quantitative polymerase chain reaction (qPCR). Protein levels of Bax, Bcl-2, caspase-3, and phosphorylation of MAPKs were measured using Western blotting assays. The MAPK signaling pathway was blocked by pretreatment with JNK inhibitor SP600125. LPS treatment induced a significant decrease in cell metabolism, viability, and ALP activity in MC3T3-E1 cells. LPS also significantly decreased mRNA expressions of osteoblast-related genes in MC3T3-E1 cells. On the other hand, LPS significantly upregulated mRNA expressions and protein levels of Bax and caspase-3 as well as activation of caspase-3, whereas decreased Bcl-2 expression in MC3T3-E1 cells. Furthermore, LPS significantly promoted MAPK pathway including the phosphorylation of JNK and the phosphorylation of ERK1/2; moreover, pretreatment with JNK inhibitor not only attenuated both of phosphorylation-JNK and ERK1/2 enhanced by LPS in MC3T3-E1 cells, but also reversed the downregulated expressions of osteoblast-specific genes including ALP and BSP induced by LPS. In conclusion, LPS could induce osteoblast apoptosis and inhibit osteoblast differentiation via activation of JNK pathway.