B cell receptor-mediated apoptosis of human lymphocytes is associated with a new regulatory pathway of Bim isoform expression

Studies in Bim-deficient mice have shown that the proapoptotic molecule Bim plays a key role in the control of B cell homeostasis and activation. However, the role of Bim in human B lymphocyte apoptosis is unknown. We show in this study that, depending on the degree of cross-linking, B cell receptors can mediate both Bim-dependent and apparent Bim-independent apoptotic pathways. Cross-linked anti-mu Ab-mediated activation induces an original pathway governing the expression of the various Bim isoforms. This new pathway involves the following three sequential steps: 1) extracellular signal-regulated kinase-dependent phosphorylation of the BimEL isoform, which is produced in large amounts in healthy B cells; 2) proteasome-mediated degradation of phosphorylated BimEL; and 3) increased expression of the shorter apoptotic isoforms BimL and BimS.     

Activation via multiple signaling pathways induces down-regulation of platelet-activating factor receptors on human B lymphocytes

Platelet-activating factor receptor (PAFR) has been identified in B cell lines and primary human B cells, but the regulation of PAFR during B cell activation has not been completely elucidated. In the present study, we have investigated the effects of B cell activation on PAFR binding parameters, PAFR mRNA and PAF-triggered intracellular calcium mobilization. The human B lymphoid cell line LA350 was shown to exhibit high levels of PAFR (48,550 +/- 4,310 sites/cell) as determined by radio-ligand binding assay with PAFR antagonist [3H]WEB2086. Treatment with phorbol 12,13-dibutyrate caused a biphasic reduction of PAFR binding. The early phase was inhibited by the protein kinase C inhibitor bisindolylmaleimide I (BIM), whereas the late phase was not blocked by BIM, protein tyrosine kinase inhibitor genistein, or the mitogen-activated protein kinase/extracellular signal-related kinase inhibitor PD98059. However, staurosporine, a broad-spectrum protein kinase inhibitor, completely inhibited the late phase down-regulation. Ionomycin also decreased [3H]WEB2086 binding sites, whereas the combination of PDB and ionomycin induced a greater reduction than either agent alone. Cross-linking of B cell receptor by anti-IgM Ab also induced down-regulation of PAFR, which was abolished by genistein or PD98059, but not by BIM or staurosporine. The decrease in surface PAFR number was closely paralleled by the reduction in PAFR mRNA both in LA350 cells and human tonsillar B cells, and was associated with decreased response to PAF indicated by decreased intracellular calcium mobilization. These data show that multiple signaling pathways are involved in down-regulating PAFR expression during B cell activation and development.     

Interferon-induced transfer of viral resistance by human B and T lymphocytes

Enriched human B lymphocytes cocultivated with mouse L cells produced human leukocyte interferon (IFN-alpha) and shortly thereafter transferred antiviral activity to the recipient cells (99% inhibition of expected virus yield). In contrast, cocultivation of enriched T-cell populations with mouse L cells resulted in no IFN production or transfer of antiviral activity. In addition, both T and B lymphocytes pretreated with exogenous IFN or stimulated in vitro by mitogens could transfer antiviral activity to human WISH cells. The transfer of antiviral activity was not blocked by antibodies to IFN. The data indicate that both T and B cells can be recruited by IFN to transfer antiviral activity. Thus, once cells are recruited by IFN they can transfer antiviral activity in the absence of IFN and protect cells locally or distally from the site of infection.     

EBV infection renders B cells resistant to growth inhibition via adenylyl cyclase

Cyclic AMP (cAMP) is an important physiological growth inhibitor of lymphoid cells, and the cAMP/protein kinase A (PKA) pathway is disrupted in several immunological disorders and cancers. Epstein Barr virus (EBV) infection of B lymphocytes is responsible for the development of lymphoproliferative disease as well as certain B-lymphoid malignancies. Here we hypothesized that EBV infection might render B lymphocytes resistant to cAMP/PKA-mediated growth inhibition. To test this, we assessed the growth-inhibitory response of cAMP-elevating compounds such as forskolin and isoproterenol, as well as the PKA activator 8-CPT-cAMP in normal B lymphocytes, EBV-infected B cells and in the EBV-negative B lymphoid cell line Reh. We could demonstrate that EBV infection indeed abolished cAMP-mediated growth inhibition of B cells. The defect was pinpointed to defective adenylyl cyclase (AC) activation by forskolin and isoproterenol, resulting in reduced formation of cAMP and lack of PKA activation and CREB phosphorylation. In contrast, 8-CPT-cAMP which directly activates PKA was able to inhibit EBV-infected B cell growth. The physiological implications of these results were underlined by the observation that the ability of forskolin to inhibit camptothecin-induced apoptosis was abolished in EBV-infected B cells. We conclude that EBV infection of B cells abrogates the activation of AC and thereby cAMP formation, and that this dysfunction renders the cells resistant to growth inhibition via the cAMP/PKA pathway.     

Deficient TACI expression on B lymphocytes of newborn mice leads to defective Ig secretion in response to BAFF or APRIL

Capsular polysaccharides of encapsulated bacteria do not induce immune response in newborns and the mechanism for this unresponsiveness is not clear. In adults, transmembrane activator and calcium-modulator and cyclophilin [corrected] ligand interactor (TACI) is a TNFR family member molecule with a pivotal role in Ab responses against polysaccharide vaccines. We investigated the expression and the functions of the TNF family cytokines, B cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL), and their receptors in newborn mice and found that TACI expression on B lymphocytes was dramatically reduced (p < 0.0001) in newborns as compared with adults. More importantly, TACI ligands BAFF or APRIL were unable to induce IgA/IgG/IgM secretion from newborn B lymphocytes. Additionally, TACI expression seems to be important in plasma cell development. Indeed, in contrast to adults, stimulation of newborn B lymphocytes with BAFF or APRIL did not result in up-regulation of CD138 expression. In vitro or in vivo exposure of newborn B lymphocytes to oligodeoxynucleotides (CpG ODN) led to up-regulation of TACI expression on newly formed, follicular, and marginal zone as well as B1 B lymphocyte populations, and rendered them responsive to BAFF- or APRIL-mediated CD138 expression and IgA/IgG secretion. Finally, immunization of newborn BALB/c mice but not TACI knockout mice with CpG ODN containing (4-hydroxy-3-nitrophenyl)acetyl-Ficoll led to development of IgG Abs against (4-hydroxy-3-nitrophenyl)acetyl. These findings demonstrate that low TACI expression may be a critical factor that determines the susceptibility of newborns to infections with encapsulated bacteria and the impaired immunogenicity of polysaccharide vaccines. Finally, CpG ODNs may correct deficient newborn response to polysaccharide vaccines by up-regulating TACI.     

Nonrecirculating memory T lymphocytes in cellular resistance to infection

Acquired resistance of rats to intracellular infection with Listeria monocytogenes rests on the cooperation between sensitized mediator lymphocytes and effector macrophages. Large numbers of specific T lymphoblasts, capable of transferring resistance to recipients, appear in central lymph shortly (3–6 days) after subcutaneous infection of rats. In contrast, late-phase immunity is poorly transferrable with thoracic duct lymphocytes (TDL) despite high levels of specific resistance observed in spleen, liver, and testes of actively immunized animals. That late-phase immunity is mediated partly by resident, nonrecirculating T cells is attested to by the ineffective transfer of resistance from preinfected to normal partners of parabiotic rats. Transfer studies with thoracic duct cells and peritoneal cells from the stimulated and unstimulated peritoneal cavity seem to suggest that resident T cells mediating late-phase resistance are the progeny of lymphoblasts that extravasated during early phase. Assays measuring the proliferative response upon antigen stimulation in vitro support the concept of a gradual redistribution within the animal of memory T cells.     

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with ~ 45% of its normal content. ESI–MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or γ-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.     

Inducible resistance to Fas—mediated apoptosis in B cells

Apoptosis produced in B cells through Fas(APO-1,CD95) triggering is regulated by signals derived from other surface receptors:CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas-induced cell death,whereas antigen receptor engagement,or IL-4R engagement,inhibits Fas killing and in so doing induces a state of Fas-resistance,even in otherwise sensitive,CD40-stimulated targets.Surface immunoglobulin and IL-4R utilize at least partially distinct path ways to produce Fas-resistance that differentially depend on PKC and STAT6,respectively.Further,surface immunoglobulin signaling for inducible Fas-resistance bypasses Btk,requires NF-κB,and entails new macromolecular synthesis.Terminal effectors of B cell Fas-resistance include the known anti-apoptotic gene products,Bcl-XL and FLIP,and a novel anti-apoptotic gene that encodes FAIM (Fas Apoptosis Inhibitory Molecule).faim was identified by differential display and exists in two alternatively spliced forms;faim-S is broadly expressed,but faim-L expression is tissue-specific.The FAIM sequence is highly evolu tionarily conserved,suggesting an important role for this molecule throughout phylogeny.Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells,whereas autoreactive B cells fail to become Fas-resistant and are deleted via Fas-dependent cytotoxicity.Inadvertent or aberrant acquisition of Fas-resistance may permit autoreactive B cells to escape Fas deletion,and malignant lymphocytes to impede anti-tumor immunity.     

RAC3 down-regulation sensitizes human chronic myeloid leukemia cells to TRAIL-induced apoptosis

The nuclear receptor coactivator RAC3 plays important roles in many biological processes and tumorigenesis. We found that RAC3 is over-expressed in human chronic myeloid leukemia cells K562, which are normally resistant to TRAIL-induced apoptosis. RAC3 down-regulation by siRNA rendered these cells sensitive to TRAIL-induced cell death. In addition to the up-regulation of TRAIL receptors, the process involves Bid, caspases and PARP activation, loss of mitochondrial membrane potential, and release of AIF, cytochrome c and Smac/DIABLO to the cytoplasm. We conclude that RAC3 is required for TRAIL resistance and that this anti-apoptotic function is independent of its role in hormone receptor signaling.     

3H-uridine incorporation by small lymphocytes of tolerant rats: relationship to T and B lymphocytes.

Tissue tolerance was induced in neonatal rats by the intravenous injection of bone marrow cells from adult allogeneic rat donors. After 6 to 8 weeks, lymphoid cells from rats in which tolerance had been induced were tested for mixed lymphocyte reactivity (MLR), 3H-uridine uptake, and the relationship of uridine incorporation to B and T lymphocytes. Lymph node (LN) and spleen (SPL) cells from the adult inoculated rats showed no reactivity in the MLR or normal lymphocyte transfer reaction (NLTRx), indicating that the animals were tolerant. After in vitro exposure to 3H-uridine, an abundance of small lymphocytes (SL) from these same tolerant rats were heavily labeled, in contrast to nontolerant controls, where relatively few SL were heavily labeled. In order to determine whether the heavily uridine-labeled cells were T cells or B cells, lymphoid cells from the LN and SPL of tolerant animals were exposed to either rabbit anti-AKR brain serum or rabbit anti-rat Ig conjugated with ferritin. The results showed that the heavily uridine-labeled SL of the tolerant rats were mainly Ig-positive cells.     

Inhibition of polyamine synthesis in human B lymphocytes during primary infection with Epstein-Barr virus (EBV) blocks cellular DNA synthesis but not the expression of EBV-encoded nuclear antigens (EBNA)

Inhibition of polyamine synthesis by 2-difluoromethylornithine (DFMO) treatment had no apparent effect on the initial manifestation of Epstein-Barr virus (EBV) infection in human B lymphocytes, because the expression of EBV-encoded nuclear antigens (EBNA) occurred normally. However, many subsequent steps in the transformation process were inhibited by DFMO treatment. These include cellular DNA synthesis and immunoglobulin (IgM, IgG and IgA) synthesis and secretion. Consequently, DFMO treatment blocked the progression of the transformation process of EBV-infected B lymphocytes. EBV-carrying marmoset B lymphocytes (B95-8 cells) were also blocked in their DNA synthesis when treated with DFMO. At variance with other DNA synthesis inhibitors, which induce virus production very effectively in B95-8 cells, DFMO caused no increase in the number of cells expressing the early antigens associated with the lytic cycle.     

Ectopic expression of a UDP-glucose:phenylpropanoid glucosyltransferase leads to increased resistance of transgenic tobacco plants against infection with Potato Virus Y

Transgenic tobacco plants over-expressing a salicylate- and pathogen-inducible glucosyltransferase (TOGT) acting on various phenylpropanoids show enhanced resistance against infection with potato virus Y (PVY). The transgenic plants are characterized by a several-fold increased glucosyltransferase activity in leaves as well as in roots. Under non-infectious conditions profiles of phenylpropanoids in leaves of transgenic lines were similar to that of controls. Feeding experiments with leaf-discs demonstrated a higher capacity for glucosylation of the coumarin scopoletin. After inoculation with PVY the transgenic lines showed similar formation of necrotic leaf lesions but significantly decreased levels of virus coat-protein when compared with control plants. Thus, our results imply that the activity of TOGT and the subsequent accumulation of glucosylated coumarins represent an important step in the cascade of events resulting in confinement of viral pathogens.