CpG DNA rescues B cells from apoptosis by activating NFkappaB and preventing mitochondrial membrane potential disruption via a chloroquine-sensitive pathway

Isolated murine splenic B cells gradually undergo spontaneous apoptosis while WEHI-231 B lymphoma cells undergo activation-induced apoptosis. Unmethylated CpG dinucleotides in a particular sequence context (CpG motif) in bacterial DNA or in synthetic oligodeoxynucleotides (CpG DNA) rescue both splenic B cells and WEHI-231 cells from apoptosis, an effect which could potentially contribute to autoimmune disease. Chloroquine has been used as an effective therapeutic agent for some autoimmune diseases, although the mechanism of action is not clearly understood. Low concentrations of chloroquine (<5 microM) selectively abolished CpG DNA-mediated protection against spontaneous apoptosis of splenic B cells and against anti-IgM-induced apoptosis of WEHI-231 cells without affecting anti-apoptotic activities of anti-CD40 or lipopolsaccharide. CpG DNA effectively prevented mitochondrial membrane potential disruption through a chloroquine-sensitive pathway in splenic B cells. Apoptosis protection by CpG DNA was also associated with increased expression of several proto-oncogenes and oncoproteins directly and/or indirectly through a rapid and sustained activation of NFkappaB in splenic B cells and WEHI-231 cells. These effects were also suppressed by chloroquine. Our results suggest that despite the difference in maturation phenotype of splenic B cells and WEHI-231 cells, CpG DNA rescues both from apoptosis by similar pathway, which is blocked at an early step by chloroquine.     

Reg2 protects mouse insulinoma cells from streptozotocin-induced mitochondrial disruption and apoptosis

We reported previously that pancreas-specific ablation of IGF-I in mice induced an increased expression of regenerating family proteins Reg2 and Reg3β in the pancreas and protected them from streptozotocin (Stz)-induced β-cell damage. We, therefore, assessed the effect of ectopically introduced Reg2 on Stz-induced apoptosis in MIN6 mouse insulinoma cells and report here that Reg2 protects MIN6 cells from Stz-induced apoptosis by attenuating its ability to disrupt mitochondrial membrane integrity, activate caspase-3 and promote poly-ADP ribose polymerase cleavage, and induce apoptosis. These changes correlated with suppression of c-jun N-terminal kinase (JNK) phosphorylation by Stz. Reg2 inhibited Stz-induced proapoptotic events as well as the inactivation of JNK. Inclusion of chemical inhibitor of JNK to Reg2 expressing cells rendered them sensitive to Stz. These data demonstrate that Reg2 protects insulin-producing cells against Stz-induced apoptosis by interfering with its cytotoxic signaling upstream of the intrinsic proapoptotic events by preventing its ability to inactivate JNK.     

Reg2 protects mouse insulinoma cells from streptozotocin-induced mitochondrial disruption and apoptosis

We reported previously that pancreas-specific ablation of IGF-I in mice induced an increased expression of regenerating family proteins Reg2 and Reg3β in the pancreas and protected them from streptozotocin (Stz)-induced β-cell damage. We, therefore, assessed the effect of ectopically introduced Reg2 on Stz-induced apoptosis in MIN6 mouse insulinoma cells and report here that Reg2 protects MIN6 cells from Stz-induced apoptosis by attenuating its ability to disrupt mitochondrial membrane integrity, activate caspase-3 and promote poly-ADP ribose polymerase cleavage, and induce apoptosis. These changes correlated with suppression of c-jun N-terminal kinase (JNK) phosphorylation by Stz. Reg2 inhibited Stz-induced proapoptotic events as well as the inactivation of JNK. Inclusion of chemical inhibitor of JNK to Reg2 expressing cells rendered them sensitive to Stz. These data demonstrate that Reg2 protects insulin-producing cells against Stz-induced apoptosis by interfering with its cytotoxic signaling upstream of the intrinsic proapoptotic events by preventing its ability to inactivate JNK.     

Upregulation of BNIP3 and translocation to mitochondria mediates cyanide-induced apoptosis in cortical cells

Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a Bcl-2 homology domain 3 (BH3) domain only protein, has been identified as a mitochondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary rat cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either increased with BNIP3 cDNA (BNIP3+) or knocked down with small interfering RNA (RNAi). In BNIP3+ cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (delta psim), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Ala-Asp-fluoromethyl ketone (zVAD-fmk) suppressed BNIP3+-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knockdown by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3 delta TM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3 delta TM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3+ cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of delta psim and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduce delta psim. This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death.     

Injurious effect on rat liver mitochondria by lymphocytes from patients with primary biliary cirrhosis.

Lymphocytes from primary biliary cirrhosis (PBC) patients were shown to have an injurious effect on rat liver mitochondria, as was demonstrated by the inhibition of mitochondrial respiratory control by these cells. The incubation of the PBC patients' lymphocytes with isolated rat liver mitochondria produced a significant inhibition of mitochondrial respiration in the presence of ADP. However, no significant effect on respiration was seen with control lymphocytes of normal persons or with lymphocytes from patients with alcoholic cirrhosis and miscellaneous liver diseases. The results suggest that this injurious effect of PBC lymphocytes on mitochondria might be a consequence of sensitization in vivo of the PBC patients' lymphocytes by the mitochondrial antigens.     

Delayed phytohemagglutinin-stimulated production of adenosine triphosphate by aged human lymphocytes: Possible relation to mitochondrial dysfunction

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alterations in lymphocytes from older animals. We measured levels of lymphocyte adenosine triphosphate (ATP) and continuous [³H]thymidine incorporation in phytohemagglutinin-stimulated lymphocytes from young and old humans, and the effects thereon of inhibitors of mitochondrial oxidative phosphorylation and protein synthesis. No difference was found in adenine nucleotide content between young and old subjects. After 24 hours of culture there was a decrease in ATP, with recovery and 2–3-fold increase at 48 hours in young cells after phytohemagglutinin stimulation. We observed a clearcut delay in older lymphocytes of the increase in ATP and [³H]thymidine incorporation following phytohemagglutinin stimulation. We found no evidence for decreased viability or diminished number of responding units in aged cultures. The evidence suggests that mitochondrial dysfunction may play a role in the immunodeficiency of aging.     

NGF rescues human B lymphocytes from anti-IgM induced apoptosis by activation of PKCzeta.

Nerve growth factor (NGF) is a neurotrophic factor acting on both the peripheral and central nervous systems. In addition, it has been shown to modulate B lymphocyte function through receptors consisting of both p75 and TrkA proteins. The low-affinity NGFR, p75, shares structural homology with the B cell antigen, CD40, tumor necrosis factor (TNF) receptor and Fas antigen (APO-1), which play a role in cell apoptosis. We studied the effect of NGF on anti-IgM-induced apoptosis in human B lymphocytes and the role of protein kinase C (PKC) in this effect. Incubation of Ramos cells with anti-IgM (10 microg/ml) induced apoptosis which was observed after 6 h and reached plateau levels after 24 h. Addition of NGF to anti-IgM-treated cells rescued cells from apoptosis. The NGF effect was blocked by anti-NGF antibody and by K252a, a specific inhibitor for the tyrosine kinase activity of TrkA. NGF induced translocation of PKCdelta and PKCalpha from the cytosol to the plasma membrane and translocation of PKCzeta to the nucleus. To examine the role of PKC in the inhibitory effect of NGF on anti-IgM-induced apoptosis, we used inhibitors of PKCalpha and PKCdelta and found that these treatments did not alter the NGF effect. In contrast, treatment of the cells with oligonucleotide antisense directed against the 5' coding sequence of PKCzeta reduced the expression of PKCzeta in the cells and abolished the protective effect of NGF on anti-IgM-induced apoptosis. The translocation of PKCzeta and the protective effect of NGF were inhibited by the phosphatidylinositol 3 (PI3)-kinase inhibitors wortmannin and LY294002. The results of this study indicate that NGF is involved in B cell survival and that this effect is mediated by PI3-kinase-dependent activation of PKCzeta.     

Mitogenic Responses to Lipopolysaccharide by B Lymphocytes from Patients with Systemic Lupus Erythematosus

Peripheral blood lymphocytes from 43 patients with systemic lupus erythcmatosus (SLE) and from age- and sex-matched normal controls were cultured with lipopolysaccharide (LPS) to examine the response to the polyclonal B-cell activator. Lymphocytes from active SLE patients incorporated 4840±471 (mean ± SE) cpm in response to LPS, whereas lymphocytes from inactive SLE patients incorporated 6906 ± 897 cpm. In contrast, lymphocytes from normal individuals incorporated 7452 ± 1126 cpm. Ig synthesis of lymphocytes from active SLE in response to LPS stimulation was also less than that of normal individuals. The helper T-cell function of active SLE, as examined by co-culturing irradiated SLE lymphocytes with unirradiated normal lymphocytes, was normal. These results thus suggested that a defect of B lymphocytes exists in active SLE patients. This B-cell defect and T suppressor cells apparently play an important role in the pathogenous of SLE.     

B lymphocytes are resistant to death receptor 5-induced apoptosis

Death Receptor 5 (DR5) induces apoptosis in various types of cells and is a potential therapeutic target. We have investigated whether targeting DR5 could be used to eliminate pathogenic B lymphocytes from systemic lupus erythematosus (SLE) patients. We examined DR5 expression and function on B lymphocytes from healthy controls subjects, SLE patients, and human tonsil. DR5 was expressed similarly on all B cell subpopulations, including resting and activated B cells. Expression of DR5 was equivalent on B cells from SLE patients and healthy subjects. Additionally, DR5 expression was unchanged after B lymphocyte stimulation. However, B cells were resistant to DR5-induced apoptosis, including after in vitro activation. No changes in subsets of B cells were observed in subjects of a trial of CS-1008, an agonist anti-DR5. While DR5 shows promise as a way to selectively eliminate tumor cells and activated synoviocytes, these data suggest DR5 alone cannot be used as a target to remove pathogenic SLE B cells.     

Lipopolysaccharide prevents apoptosis and induces responsiveness to antigen receptor cross-linking in immature B cells.

Unlike mature B cells, immature B cells are not activated in response to antigen receptor cross-linking. To examine the mechanisms underlying this unresponsiveness, we have studied the effects of reagents that have been shown to alter the responses of immature B cells to antigen receptor stimulation. Bacterial lipopolysaccharide (LPS) is a polyclonal B-cell activator, and has been shown to interfere with B-cell tolerance induction in vivo and in vitro. Here we show that LPS can also overcome the unresponsiveness of immature B cells to stimulation with anti-receptor (anti-mu) antibodies. LPS synergizes with anti-mu to induce a proliferative response that exceeds the response of immature B cells to LPS alone. Moreover, pretreatment of immature cells with LPS allows them to proliferate in response to subsequent stimulation with anti-mu antibodies. This induction of responsiveness to anti-mu requires exposure to LPS for at least 8 hr. Although the mechanisms of induction are not fully understood, one component of the LPS effect appears to involve enhancement of immature B-cell survival in culture. Neonatal splenic B cells undergo spontaneous apoptosis at a much higher rate than mature B cells, but we have found that LPS causes a dramatic inhibition of apoptosis, even when it is present for only the first 8 hr of culture. The ability of LPS to promote survival of immature B cells and allow them to proliferate in response to antigen receptor stimulation provides a system for investigation of the biochemical mechanisms of unresponsiveness and tolerance susceptibility.     

TNF-alpha protects human primary articular chondrocytes from nitric oxide-induced apoptosis via nuclear factor-kappaB

TNF-alpha plays a key role in rheumatoid arthritis, but its effect on chondrocyte survival is still conflicting. In the present study, we tested how TNF-alpha influences chondrocyte survival in response to nitric oxide (NO)-related apoptotic signals, which are abundant during rheumatoid arthritis. Human primary articular chondrocytes or cartilage explants were pretreated with TNF-alpha for 24 hours and then treated with the proapoptotic NO donor sodium-nitro-prusside (SNP) for an additional 24 hours. TNF-alpha pretreatment markedly protected chondrocytes from SNP-induced cell death. Preincubation of chondrocytes with TNF-alpha inhibited both SNP-induced high-molecular weight DNA fragmentation and annexin V-FITC binding. Of interest, TNF-alpha induced persistent nuclear factor-kappaB (NF-kappaB)-DNA binding activity even in the presence of SNP, mirroring apoptosis protection effects. Both the TNF-alpha antiapoptotic effect and NF-kappaB-DNA binding activity were significantly inhibited by NF-kappaB inhibitors, Bay 11-7085, MG-132, and adenovirus-expressing mutated IkappaB-alpha. Phosphatidylinositol-3 kinase inhibitor LY 294002 also markedly inhibited the antiapoptotic effect of TNF-alpha. In primary chondrocytes, TNF-alpha induced expression of the antiapoptotic protein Cox-2, which persisted in the presence of SNP, and a specific Cox-2 inhibitor significantly blocked the TNF-alpha protective effect. We therefore conclude that TNF-alpha-mediated protection of chondrocytes from NO-induced apoptosis acts through NF-kappaB and requires Cox-2 activity.     

杨梅苷通过抑制线粒体功能障碍减轻MPP~+诱导的SN4741细胞凋亡

目的:研究杨梅苷(myricitrin,五羟基黄酮-3-鼠李糖,Myr)对1-Methyl-4-phenylpyridinium iodide(MPP+)诱导的SN4741细胞凋亡的影响及其可能机制。方法:利用MPP+处理多巴胺能SN4741细胞,建立帕金森病体外细胞模型;4-甲基偶氮唑蓝(MTT)检测SN4741细胞活性;TUNEL法检测细胞凋亡断裂的DNA片段;Mitotracker观察线粒体形态;流式细胞术检测活性氧(reactive oxygen species,ROS)。结果:MPP+(80μmol/L)作用于SN4741细胞24 h后,与对照组比较,细胞存活率降低(47.3±2.9)%(P0.01),断裂DNA片段增多,线粒体碎片增多,ROS生成增多;杨梅苷(10、20μmol/L)预处理24h后,细胞存活率增加(69.3±3.2)%和(87.7±5.2)%(P0.01),DNA断裂片段减少,线粒体碎片减少,ROS生成减少。结论:杨梅苷对MPP+诱导SN4741细胞凋亡具有浓度依赖性的保护作用,其作用机制可能与减轻线粒体功能障碍、抑制ROS生成有关。     

Apoptosis of human primary B lymphocytes is inhibited by N-acetyl-L-cysteine

Thiols are important molecules to control apoptosis. This study examined the effect of N-acetyl-L-cysteine (NAC) on in vitro spontaneous apoptosis of human tonsillar B lymphocytes (TBL). Results show that NAC inhibits TBL apoptosis and maintains their survival in vitro. The antiapoptotic action of NAC is progressively reduced when its addition to culture is delayed, is reversible, and is not blocked by cycloheximide. The antiapoptotic activity of NAC is associated with its ability to inhibit caspase-3 and -7 proteolytic processing, DNA-fragmentation factor 45 cleavage, and DNA fragmentation. Furthermore, NAC inhibits BID cleavage and cytochrome c release from mitochondria and increases the expression of Bcl-2 and Bcl(XL) survival proteins. However, it has no effect on caspase-9 cleavage and increases that of caspase-8 and poly(adenosine 5'-diphosphate-ribose)polymerase. We conclude that NAC-induced inhibition of TBL apoptosis is associated with inhibition of caspase-3 and -7 processing and is accompanied by changes in several regulatory components of the apoptotic process. These results pose the question of whether microenvironment thiols may in part contribute to in vivo B cell survival.     

Increased susceptibility of cord blood B lymphocytes to undergo spontaneous apoptosis

In this study, we compared the rate of spontaneous apoptosis of B cells from umbilical cord blood with adult B cells and assessed the role of Bcl-2, CD5, interleukin (IL)-4 and B cell-activating factor in B cell spontaneous apoptosis. We found that spontaneous apoptosis of cultured B cells, as assessed by utilizing annexin-V binding, was significantly higher in cord blood than in healthy adult individuals (77.5; 95 CI, 73.5-81.5 versus 59.2; 95 CI, 54-64, respectively, P < 0.0001) and further confirmed by 4' 6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining. Whereas the expression of B cell-activating factor from the tumour necrosis factor family (BAFF) receptor mRNA was similar in B cells from adults and cord blood, we detected lower levels of circulating BAFF in the serum of cord blood (0.68 +/- 0.13 ng versus 1.83 +/- 0.54 ng, P = 0.01). The latter may explain, in part, our observation of lower levels of mean fluorescence intensity of Bcl-2 in cord B cells compared with adults (1.6 +/- 0.9 versus 2.85 +/- 1.3, P = 0.033). CD19(+) CD5(+) B cells from cord blood underwent a lower rate of apoptosis in comparison to CD19(+) CD5(-) B cells (25.1 +/- 9.3%versus 58.5 +/- 12.5%, P < 0.0001). This pattern of sensitivity was comparable in adult blood (15 +/- 5.5%versus 22.7 +/- 9.3%, P = 0.01). Nevertheless, the rate of apoptosis was higher in CD19(+) CD5(+) from cord blood compared to CD19(+) CD5(+) from adults (25.1 +/- 9.3%versus 15 +/- 5.5%, P = 0.0013). The addition of rIL-4 (10 u/ml) to cultured cells decreased B cell apoptosis in a similar fashion in both cord and adults blood. This rescue was strengthened when BAFF (100 microg/ml) was further added. Thus, alterations in Bcl-2 or serum BAFF level may explain the increased rate of cord blood B cell apoptosis.     

An X;9 translocation, primary amenorrhea, and hypothalamic dysfunction

A white girl presented at age 16 yr with delayed puberty and primary amenorrhea. She had 46 chromosomes with a de novo reciprocal X;9 translocation. The normal X chromosome was found to be heterochromatic, thus preserving the function of the translocation portion of the 9. Her total estrogen and serum estradiol levels were low and her serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were at the lower end of the normal adult range. She had a reasonably good FSH and LH response to GnRH, but an inadequate response to 100 mg of clomiphene daily for 1 wk. This would suggest that the abnormality of function is probably hypothalamic, a hitherto unreported association. De novo translocations between X chromosomes and autosomes are rare and none identical to this case has been described. The breakpoint of the X chromosome was at p22, well outside the "critical region" for female reproductive function. It seems probable that her chromosome abnormality is responsible for her clinical state.     

肝细胞生长因子拮抗放线菌素D诱导的肝细胞凋亡及机制探讨

目的： 探讨肝细胞生长因子（HGF）对放线菌素D (ActD)诱导HL7702肝细胞凋亡的拮抗作用及可能机制。 方法： 本实验采用HL7702正常人肝细胞株，MTT法检测ActD对肝细胞存活力的影响；Hoechst33342进行凋亡形态学染色；DNA凝胶电泳及流式细胞仪检测细胞凋亡数量；Western blotting方法检测细胞总Akt及磷酸化Akt蛋白的表达。 结果： ActD可以诱导HL7702肝细胞凋亡，其浓度在0.25-8 mg/L范围内呈现剂量效应关系；PI3K特异性抑制剂wortmannin能够增强ActD诱导的肝细胞凋亡作用；肝细胞生长因子(HGF)对ActD诱导的肝细胞凋亡有拮抗作用，在一定剂量范围内呈现剂量效应关系；并且HGF能够激活PI3K/Akt信号转导途径；进一步用wortmannin阻断PI3K/Akt信号途径后，HGF的拮抗凋亡作用被抑制。 结论： 一定剂量的ActD可以诱导肝细胞凋亡；wortmannin能够增强ActD诱导肝细胞凋亡的作用；HGF对ActD诱导的这种细胞凋亡有明显的拮抗作用，并且HGF的抗凋亡作用与其激活细胞内PI3K/Akt信号转导通路有关。     

Control of primary IgM antibody responses to H-2 alloantigens by antigen-bearing live B lymphocytes

Alloantigen-bearing (H-2d+) peripheral red blood cells, but not red cell-depleted H-2d+ spleen cells, induce primary IgM anti-H-2d plaque-forming cell responses. In this study it is reported that the primary antibody responses to H-2d+ peripheral red blood cells can be markedly suppressed by a subpopulation of H-2d+ spleen cells when they are injected simultaneously or a few days before injection of red blood cells. This suppression was antigen (H-2d)-specific, did not depend on T cells of either the donor or the recipient, and strictly required live donor cells. An energy-dependent action of the donor cell cortex and some proliferation of donor cells in the recipient seemed to be involved in the mechanism of suppression. The donor-suppressor cell type was largely present in the spleen but not in the bone marrow and thymus, and was present in the spleen of athymic nude mice. The suppressor cells displayed the properties of B lymphocytes: they adhered to the nylon wool but not to glass, were of relatively low density (rho less than 1.09), and were surface Ig+, Ia+, Fc receptor-positive but Thy-1-. H-2d+ suppressor-donor B lymphocytes might directly signal to antigen-specific recipient B cells competing with the signal provided by H-2d+ red blood cells for the B cell activation.