CD137-induced apoptosis is independent of CD95

CD95 (APO-1/Fas) and CD137 (ILA/4-1BB) are members of the tumour necrosis factor receptor family, and both are involved in induction of apoptosis in lymphocytes. Contrary to the case of CD95, apoptosis by CD137 is caused by cross-linking of the respective ligand rather than the receptor. Nothing is known so far about the mechanism of CD137-induced cell death. Here, we show that immobilized CD137 protein induces expression of CD95 in resting primary T and B lymphocytes. However, induction of apoptosis by CD137 is independent of CD95, because: (1) antagonistic anti-CD95 antibody fragments do not block CD137-induced apoptosis; and (2) CD137, but not anti-CD95, can induce apoptosis in resting lymphocytes.     

The CD40-induced protection against CD95-mediated apoptosis is associated with a rapid upregulation of anti-apoptotic c-FLIP

CD95/Fas and CD40 receptors are important regulators of cell survival during germinal center reaction. In this study we used a human follicular lymphoma cell line, HF1A3, to study molecular mechanisms of CD95-mediated apoptosis and CD40-induced rescue from apoptosis. CD95 stimulation induced activation of caspase-8 and -3, collapse of mitochondrial membrane potential (DeltaPsim), release of cytochrome c and fragmentation of nuclear DNA. All these apoptotic events were abrogated, when cells were pretreated with CD40 antibodies before CD95 stimulation. CD40 induced a rapid up regulation of both short and long isoforms of c-FLIP, as these proteins were detectable 4h after receptor stimulation. The induction of c-FLIP as well as the anti-apoptotic function of CD40 was completely abolished when NF-kappaB activity was inhibited by a selective inhibitor PDTC. We conclude that the anti-apoptotic signaling of CD40 involves NF-kappaB-mediated induction of c-FLIP proteins which can interfere with caspase-8 activation. However, it remains to be seen whether c-FLIP proteins are the only one ones involved in CD40-mediated protection.     

Rapid CD40-mediated rescue from CD95-induced apoptosis requires TNFR-associated factor-6 and PI3K

The activation molecule CD40 and the death receptor CD95/Fas play important roles in regulating B cells so that effective antimicrobial immunity occurs without autoimmunity. CD40 signaling increases CD95 expression, sensitizing cells to apoptosis, but sustained CD40 signals rescue B cells from CD95 killing. Here we describe a mechanism of early CD40-mediated rescue from CD95-induced apoptosis in B cells. Maximal rescue was achieved when CD40 signals were given within 1-2 h of initiating CD95 apoptosis. CD40 signaling did not block association of Fas-associated death domain-containing protein with CD95, but decreased CD95-induced activation of caspases 3 and 8. Rapid CD40 rescue did not require NF-kappaB activation and was independent of de novo protein synthesis, but was dependent upon active PI3 K. Signaling via a CD40 mutant that does not bind TNFR-associated factor (TRAF)1, TRAF2, and TRAF3 rescued B cells from CD95-induced apoptosis. TRAF1/2/3-independent rescue was confirmed in B cell lines made deficient in these TRAF molecules by gene targeting. In contrast, CD40 rescue was completely abrogated in TRAF6-deficient B cells, which showed reduced activation of Akt in response to CD40 engagement. These results reveal a new rapid mechanism to balance B cell activation and apoptosis.     

Protection of CD95-mediated apoptosis by activation of phosphatidylinositide 3-kinase and protein kinase B

Apoptosis may be triggered, in a variety of tissues, by interaction of the cell surface molecule CD95 with its specific ligand, CD95L. CD95 plays a physiological role in the regulation of the immune response; furthermore, alterations in CD95/CD95L function may contribute to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases and viral infections. Many cells that express CD95, however, are not susceptible to CD95-mediated apoptosis. It is therefore important to identify the mechanisms that counteract the CD95 apoptotic process that are still poorly understood. Growth factors and lymphokines such as interleukin (IL)-4 that counteract CD95-mediated apoptosis may activate phosphatidylinositide 3-kinase (PI 3-kinase). We therefore used two different approaches to investigate the role of PI 3-kinase on CD95-mediated apoptosis. First we tested the effect of two pharmacological PI 3-kinase inhibitors, wortmannin and LY294002, on CD95 agonistic antibody-induced apoptosis in three different cell lines. Second, we co-expressed in COS7 cells CD95 with constitutively active PI 3-kinase. Results of both approaches indicate that active PI 3-kinase effectively protects against CD95-mediated apoptosis. Furthermore we extended our studies on the CD95 downstream mediator, FADD, and on the PI 3-kinase downstream mediator, the serine/threonine protein kinase PKB, using the co-expression approach in COS7 cells. We provide evidence that apoptosis induced by triggering the CD95 cell death receptor is counteracted by PI 3-kinase activation; moreover, PKB but not p70S6K represents the relevant downstream target of PI 3-kinase signaling.     

Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis

Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression.     

Protection against pneumococcal infection by a ribosomal preparation

A subcellular extract prepared from nonencapsulated Diplococcus pneumoniae type 3 protected mice against a subsequent challenge with virulent D. pneumoniae types 1, 2, 3, and 7. Potency ratios ranged from 25 (type 3) to >1,175 (type 1). The immunity induced in mice by this preparation was best obtained by intraperitoneal inoculation followed by intravenous challenge. Mice immunized in this manner were protected up to 12 weeks and could withstand a challenge of several hundred LD₅₀. The protection was destroyed by treatment of the preparation with ribonuclease and was decreased by treatment with protease. The preparation consisted of 60.5% ribonucleic acid, 29.1% protein, 6.3% deoxyribonucleic acid, and 4.0% hexose. Ultracentrifugation studies indicated that this extract had five constituents which are compatible with ribosomal material.     

Monochloramine enhances Fas (APO-1/CD95)-induced apoptosis in Jurkat T cells

Monochloramine derivatives are physiological oxidants produced by activated neutrophils. We report the effects of chemically prepared monochloramine (NH2Cl) on Fas-induced apoptosis in Jurkat T cells. When the cells were pretreated with NH2Cl (20-70 microM), subsequent addition of apoptosis-inducing anti-Fas antibody resulted in a synergistic enhancement of apoptosis. Treatment of NH2Cl (50-70 microM) alone resulted in a slight but definite apoptosis. Caspase activities, as measured by DEVD and IETD cleavage activities, were also elevated synergistically by NH2Cl + anti-Fas antibody stimulation. Moreover, a broad caspase inhibitor, Z-VAD-fmk, almost completely inhibited the apoptosis induced by NH2Cl and/or anti-Fas antibody. Fas expression on the Jurkat cell surface was not affected by the NH2Cl treatment. After 3 h of NH2Cl treatment, when the apoptosis was beginning to increase, the cells showed cytochrome c release from mitochondria, proteolytic activation of caspase 9, and poly (ADP-ribose) polymerase cleavage, regardless of Fas stimulation. Z-VAD-fmk almost completely inhibited this poly (ADP-ribose) polymerase cleavage, but not cytochrome c release. By contrast, Fas stimulation alone resulted in neither cytochrome c release nor caspase 9 activation at 3 h, and the increase in the DEVD cleavage activity and apoptosis became evident at later time points. These results suggested that NH2Cl enhanced Fas-induced apoptosis through the cytochrome c release and caspase 9 activation at the early stage of apoptosis. Chloramines derived from acute inflammation may modify immune reactions, such as cell-mediated cytotoxicity and some autoimmune diseases, by the enhancement of Fas-induced apoptosis.     

P-glycoprotein decreases with T cell maturation but is not responsible for resistance to CD95-induced apoptosis

P-glycoprotein (Pgp) is a membrane transporter responsible for resistance to chemotherapy in cancer cells. Its presence in T cells is very well documented, but its function in the immune system is still poorly understood. Recent findings suggest that Pgp may be involved in regulating programmed cell death by inhibiting caspase 8 and caspase 3. Utilising antigenically-activated T cells and the physiologically relevant apoptotic ligand, membrane CD95-L, we have previously reported that while T cells are generally resistant to CD95-induced death at early stages of activation, their susceptibility to apoptosis increases with successive activation and clonal expansion. In this study we investigated whether changes in apoptotic susceptibility were related to T cell Pgp function. Results showed that Pgp expression and function in T cells decreases with maturation, with CD8 cells having the highest Pgp function. However, although Pgp function inversely correlated with caspase 3 activity, no difference was observed between apoptotic susceptible CD25- cells and resistant CD25+ cells. In addition sorting of cells with high and low Pgp function showed no correlation with apoptotic capability. Therefore, whilst Pgp modulates caspase activity, it is not responsible for resistance to apoptosis of early activated T cells nor the increased susceptibility observed at the later stages of maturation in antigenically activated cells.     

Induction of cross-serovar protection against genital chlamydial infection by a targeted multisubunit vaccination approach

An important consideration for antichlamydial vaccine development is the induction of cross-serovar protection, since multiple serovars (D to L) of Chlamydia trachomatis cause genital infections. We have shown previously that vaccination with C. trachomatis-derived recombinant chlamydial protease-like activity factor (rCPAF) induced significant earlier resolution of Chlamydia muridarum infection and reduced oviduct pathology. However, the vaccinated mice continued to shed chlamydiae for up to 2 weeks after challenge. In this study, C. trachomatis serovar D recombinant proteins, such as recombinant major outer membrane protein (rMOMP), recombinant inclusion membrane protein A (rIncA), and rCPAF were administered intranasally, individually or in combinations, with murine interleukin-12 (IL-12) as an adjuvant, and cross-species immunity against intravaginal C. muridarum infection was examined. Immunization with rCPAF plus IL-12 (rCPAF+IL-12), compared to immunization with rIncA+IL-12 or rMOMP+IL-12, induced the greatest antigen-specific gamma interferon production from purified CD4⁺ T cells and concurrently enhanced serum antibody production. All (100%) the animals vaccinated with rCPAF+IL-12 alone or in any combination completely resolved the infection by day 18 after challenge compared to animals vaccinated with rIncA+IL-12 (50%), rMOMP+IL-12 (33%), or phosphate-buffered saline (mock vaccinated; 0%). Moreover, oviduct pathology in mice vaccinated by any regimen that included rCPAF, but not rMOMP+IL-12 or rIncA+IL-12 alone, was markedly reduced compared to mock-immunized animals. The addition of rMOMP and/or rIncA did not significantly enhance the rCPAF+IL-12-induced effect on bacterial clearance or oviduct pathology. These results suggest a greater conservation of protective linear antigenic epitopes within CPAF than MOMP or IncA across the examined serovars and the need to identify other highly conserved antigens for use with rCPAF in a multisubunit recombinant vaccine.     

Molecular basis of rifampicin-induced inhibition of anti-CD95-induced apoptosis of peripheral blood T lymphocytes: the role of CD95 ligand and FLIPs

Rifampicin modulates immune response; however, mechanisms by which it exerts these effects are incompletely understood. Recently, rifampicin has been shown to bind to and activate glucocorticoid receptors. Because of the evidence for a role of glucocorticoids in lymphocyte apoptosis, we hypothesized that rifampicin may exert its influence on the immune system by regulating apoptosis. Therefore, we examined the effect of rifampicin on signaling pathway of anti-CD95-induced apoptosis in peripheral blood lymphocytes. Rifampicin, in a concentration-dependent manner, inhibited anti-CD95-induced apoptosis in both CD4⁺ and CD8⁺ T cells, which was associated with the inhibition of activation of both caspase-3 and caspase-8. In addition, rifampicin down-regulated the expression of CD95L and Bax. The inhibitory effects of rifampicin on apoptosis and caspase activation as well as its effect on the expression of CD95L and FLIPs were reversed by RU486, an antagonist of glucocorticoid receptor. These data suggest that rifampicin inhibits anti-CD95-mediated apoptosis in lymphocytes by modulating the expression of certain proteins that regulate apoptosis, at least in part, via glucocorticoid receptors.     

Mycoplasma alligatoris infection promotes CD95 (FasR) expression and apoptosis of primary cardiac fibroblasts

Mycoplasma alligatoris causes acute lethal primary infection of susceptible hosts. A genome survey implicated sialidase and hyaluronidase, potential promoters of CD95-mediated eukaryotic cell death, as virulence factors of M. alligatoris. We used immunofluorescence imaging and flow cytometry to examine the effects of M. alligatoris infection in vitro on CD95 expression and apoptosis by alligator cardiac fibroblasts, a major cell type of a target organ of M. alligatoris infection in vivo. A uniform distribution of CD95 in primary cultured cardiac, skeletal muscle, and embryonic fibroblasts was demonstrated by using polyclonal antibodies against the N or C terminus of mouse or human CD95. Anti-CD95 antibodies reacted on Western blots of fibroblast lysates with a band with the predicted apparent molecular weight of CD95, but soluble CD95 was not detected in plasma from control or M. alligatoris-infected alligators. The proportion of CD95-gated cardiac fibroblasts increased threefold (P<0.01) 48 h after inoculation with M. alligatoris. Infection induced morphological changes in cardiac fibroblasts, including translocation of CD95 characteristic of apoptosis and an eightfold increase (P<0.16) in 5-bromo-2'-deoxyuridine (BrdU) incorporation measured in a terminal deoxynucleotide transferase dUTP nick end-labeling apoptosis assay. The proportion of BrdU-gated controls activated with agonistic immunoglobulin M against human CD95 also increased threefold (P<0.03 for muscle). Heat-inactivated M. alligatoris and sterile M. alligatoris-conditioned culture supernatant had no effect. This is the first report of a CD95 homolog in the class Reptilia and establishes a new model that can be used to test the direct bacterial interaction with upstream components of the CD95 signal transduction pathway.     

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。     

Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.     

Induction of CD95 upregulation does not render chronic lymphocytic leukemia B-cells susceptible to CD95-mediated apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of long-lived and well-differentiated clonal B-lymphocytes in peripheral blood, lymphoid tissue and bone marrow. Although B-CLL pathogenesis is not entirely understood, the progressive increase in lymphocyte counts coupled with the very low proportion of proliferating cells suggests that B-CLL may be primarily determined by defective apoptosis. Consistently, freshly analyzed CLL B-cells express very low levels of membrane CD95, one of the best-known receptors involved in triggering apoptosis. In this study, CD95 upregulation on CLL B-cells was induced by culturing clonal B-cells in the presence of supernatants from preactivated autologous T-lymphocytes. Intracellular cytokine staining of preactivated autologous T-lymphocytes using monoclonal antibodies (moAbs) specific for Th1 or Th2 cytokines, namely interleukin (IL)-2, IL-4, IL-5, IL-10 and interferon (IFN)-gamma, showed these cells to be positive for IL-2 and IFN-gamma. Blocking experiments using moAbs specific for IL-2 and/or IFN-gamma revealed that CD95 upregulation on CLL B-cells was mainly driven by IFN-gamma. However, CD95-expressing CLL B-cells were demonstrated to be resistant to CD95-mediated apoptosis, thus arguing against strategies aimed at exploiting CD95-mediated apoptosis for immunotherapy of B-CLL.     

Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation

Puerarin, a main isoflavone glycoside distributed in Pueraria lobata (Willd.) Ohwi, showed inhibitory activity on H2O2-induced PC12 cells damage in our previous work. However, there is insufficient evidence in protective mechanism of puerarin, especially that relating to the mitochondrial function. In this study, when cells were pretreated with puerarin prior to 0.4 mM MPP+, protective roles were accompanied by a reduction of cell viability loss, morphological changes of apoptosis and apoptotic rate. To explore the protective mechanism of puerarin in MPP+-induced PC12 cells, mitochondrial function and caspase-3-like activity were measured. The results indicated that puerarin inhibited the release of mitochondrial cytochrome c to cytosol and the loss of mitochondrial membrane potentials. In addition, puerarin also reduced MPP+-induced caspase-3-like activation. Taken together, the above results suggest that pretreatment of PC12 cells with puerarin could block MPP+-mediated apoptosis by mitochondria-dependent caspase cascade.     

Protection against Gram-negative infection by 'super-active' antigen.

'Super-active' antigens modified antigens released from bacteria which had been phagocytosed and killed by human leucocytes, were found to induce protective responses in mice within 24 h of immunization. At the earliest time (24 h) when immunized mice were protected against lethal intrapertoneal (i.p.) challenge by the bacteria from which which the 'super-active' antigens were made (Proteus mirabilis) the leucocytes of peripheral blood from immunized mice showed enhanced phagocytosis and killing of autologous bacteria and there was an increase in the number of lymphocytes producing anti-proteus antibody. Another mouse protective factor inducing transient protection lasting 1-2 days against lethal i.p. challenge by P. mirabilis was found in preparations of lysed heman leucocytes not engaged in phagocytosis. Burned mice, immunized with 'super-active' antigen preparations were protected against lethal invasive proteus infection, inoculated on to the burn surface, 2 h after burning and immunization.     

Protection against Yersinia infection induced by non-virulence-plasmid-encoded antigens

Specific immunity against Yersinia was induced by plasmid-encoded antigens not associated with virulence. Mice were immunised with viable bacteria from a virulence-plasmid-cured strain of Y. pseudotuberculosis. This antigenic stimulation generated specific protection against virulence-plasmid-harbouring strains of Y. pseudotuberculosis and Y. pestis, demonstrating that protection can be generated by organisms lacking plasmid-encoded virulence antigens.     

Bcl-2-mediated regulation of CD69-induced apoptosis of human eosinophils: identification and characterization of a novel receptor-induced mechanism and relationship to CD95-transduced signalling

Elimination of the eosinophils from the airways by selective induction of apoptosis represents a therapeutic approach for asthma. Here we report on a possible target molecule, the surface receptor CD69. To simulate an asthmatic response, segmental allergen challenge in mild asthmatics was performed. Eosinophil numbers increased in bronchoalveolar lavage (BAL) at 18 h. In contrast to blood cells, BAL eosinophils expressed the activation marker CD69. Purified blood eosinophils stimulated with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma) expressed CD69 and showed prolonged viability. Only IFN-gamma enhanced constitutive CD95 expression. Coincubation with anti-CD69 or anti-CD95 monoclonal antibody (MoAb) induced apoptosis, as revealed by propidium iodide incorporation, membrane blebbing and nuclear fragmentation. Additionally, both anti-CD69 and anti-CD95 MoAb reduced cytokine-enhanced Bcl-2 expression. In conclusion, CD69 transduces a Bcl-2-dependent death signal when ligated by a specific antibody. As, in contrast to the ubiquitous death-inducer CD95, the function of CD69 appears to be restricted to activated eosinophils, it represents an ideal target for therapeutic intervention in asthma.