Controlling NF-kappaB activation in T cells by costimulatory receptors

Full and productive activation of T lymphocytes relies on the simultaneous delivery of T cell receptor (TCR)- and coreceptor-derived signals. In na?ve T cells engagement of the TCR alone causes anergy, while TCR triggering of preactivated T cells results in activation-induced cell death. Costimulatory signals are prominently mirrored by the activation of NF-kappaB, which needs input from the TCR as well as from coreceptors in order to be fully activated and to fulfil its crucial function in the immune response. Coreceptor-generated signals tightly control the duration and amplitude of the NF-kappaB response. The activation of IkappaB kinase (IKK) complex at the contact zone between a T cell and an antigen-presenting cell offers the unique opportunity to study the spatial organization of IKK activation. Recent studies indicate that coreceptor pathways influence the threshold activities of many signalling mediators and thus act on multiple layers of the NF-kappaB pathway.     

Microenvironment produced by acute myeloid leukemia cells prevents T cell activation and proliferation by inhibition of NF-kappaB, c-Myc, and pRb pathways.

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-beta, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-kappaB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G(0) to G(1). Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

Mechanisms of the TRIF-induced interferon-stimulated response element and NF-kappaB activation and apoptosis pathways

Toll-like receptor-3 is critically involved in host defense against viruses through induction of type I interferons (IFNs). Recent studies suggest that a Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) and two protein kinases (TANK-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-epsilon) are critically involved in Toll-like receptor-3-mediated IFN-beta production through activation of IFN regulatory factor (IRF)-3 and IRF-7. In this study, we demonstrate that TRIF interacts with both IRF-7 and IRF-3. In addition to TBK1 and IKKepsilon, our results indicate that IKKbeta can also phosphorylate IRF-3 and activate the IFN-stimulated response element. TRIF-induced IRF-3 and IRF-7 activation was mediated by TBK1 and its downstream kinases IKKbeta and IKKepsilon. TRIF induced NF-kappaB activation through an IKKbeta- and tumor necrosis factor receptor-associated factor-6-dependent (but not TBK1- and IKKepsilon-dependent) pathway. In addition, TRIF also induced apoptosis through a RIP/FADD/caspase-8-dependent and mitochondrion-independent pathway. Furthermore, our results suggest that the TRIF-induced IFN-stimulated response element and NF-kappaB activation and apoptosis pathways are uncoupled and provide a molecular explanation for the divergent effects induced by the adapter protein TRIF.     

TIRAP mediates endotoxin-induced NF-kappaB activation and apoptosis in endothelial cells

Bacterial lipopolysaccharide (LPS) initiates multiple signaling events in vascular endothelial cells that can result in activation and/or cell death. LPS-induced activation of endothelial cells elicits a wide array of vascular endothelial responses, many of which are dependent on NF-kappaB activation. Several of the signaling molecules that mediate LPS-induced NF-kappaB activation, including Tlr-4, MyD88, and IRAK-1, have been similarly reported to mediate LPS pro-apoptotic signaling. Recently, a new signaling molecule, TIRAP, has been identified that mediates LPS-induced NF-kappaB signaling in monocytes and macrophages. Using a TIRAP dominant negative construct, we have identified a role for TIRAP in mediating LPS-induced NF-kappaB activation and apoptosis in human endothelial cells. These data identify TIRAP as a dual functioning signaling molecule and suggest the presence of a MyD88-independent LPS signaling pathway in human endothelial cells.     

Aspirin prevents apoptosis and NF-kappaB activation induced by H2O2 in hela cells

The classical pathway of nuclear factor-kappa B (NF-kappaB) activation by several inducers mainly involves the phosphorylation of IkappaBalpha by a signalsome complex composed of IkappaBalpha kinases (IKKalpha and IKKbeta). However, in some cell types hydrogen peroxide (H2O2) has been shown to activate an alternative pathway that does not involve the classical signalsome activation process. In this study, we demonstrate that H2O2 induced NF-kappaB activation in HeLa cells through phosphorylation and degradation of IkappaB proteins as shown by immunblot analysis. Our studies reveal that a commonly used non-steroid anti-inflammatory drug, acetylsalicylic acid (aspirin) prevents H2O2-induced NF-kappaB activation in a dose-dependent manner through inhibition of phosphorylation and degradation of IkappaBalpha and IkappaBbeta. Differential staining and DNA fragmentation analysis also show that aspirin preloading of HeLa cells also prevents H2O2-induced apoptosis in a dose-dependent manner with maximum efficiency at 10 mM concentration. Additionally, aspirin effectively prevents caspase-3 and caspase-9 (cysteinyl aspartate-specific proteases) activation by H2O2. These results suggest that NF-kappaB activation is involved in H2O2-induced apoptosis and aspirin may inhibit both processes simultaneously.     

Anti-viral strategies of cytotoxic T lymphocytes are manifested through a variety of granule-bound pathways of apoptosis induction

Cytotoxic T cells and natural killer cells together constitute a major defence against virus infection, through their ability to induce apoptotic death in infected cells. These cytolytic lymphocytes kill their targets through two principal mechanisms, and one of these, granule exocytosis, is essential for an effective in vivo immune response against many viruses. In recent years, the authors and other investigators have identified several distinct mechanisms that can induce death in a targeted cell. In the present article, it is postulated that the reason for this redundancy of lethal mechanisms is to deal with the array of anti-apoptotic molecules elaborated by viruses to extend the life of infected cells. The fate of such a cell therefore reflects the balance of pro-apoptotic (immune) and anti-apoptotic (viral) strategies that have developed over eons of evolutionary time.     

Plantainoside D protects adriamycin-induced apoptosis in H9c2 cardiac muscle cells via the inhibition of ROS generation and NF-kappaB activation

Plantainoside D (PD), was isolated from the leaves of Picrorhiza scrophulariiflora (Scrophulariaceae). The anti-oxidative activity of PD was evaluated based on scavenging effects on hydroxyl radicals and superoxide anion radicals. Adriamycin (ADR) is a potent anti-tumor drug known to cause severe cardiotoxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity has not been understood. This study was undertaken to investigate the protective effect of PD against ADR-induced apoptosis. In vitro, ADR caused dose-dependent toxicity in H9c2 cardiac muscle cells. Pre-treatment of the cardiac muscle cells with PD significantly reduced ADR-induced apoptosis of cardiac muscle cells. PD inhibited the ROS produced by ADR in the cardiac muscle cells. As well, PD increased GSH(glutathione), compared with ADR. In response to ADR, NF-kappaB was activated in H9c2 cells. However the treatment of PD reduced the activation of NF-kappaB. We also observed that the NF-kappaB inhibitor, PDTC, inhibited the cytotoxic effect on ADR-induced apoptosis in cardiac muscle cells. In parallel, IkappaBalpha-dominant negative plasmid-overexpression abrogated ADR-induced apoptosis in H9c2 cardiac muscle cells. In conclusion, these results suggest that Plantaionoside D can inhibit ADR-induced apoptosis in H9C2 cardiac muscle cells via inhibition of ROS generation and NF-kappaB activation. The pure compound PD can be a potential candidate agent which protects cardiotoxicity in ADR-exposed patients.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

Enhancement of NF-kappaB activation in lymphocytes prevents T cell apoptosis and improves survival in murine sepsis

Sepsis induces extensive lymphocyte apoptosis that contributes to immunosuppression and mortality. Activation of the canonical NF-kappaB pathway, however, prevents TNF-alpha-induced lymphocyte apoptosis. In this study the function of canonical NF-kappaB in T cells was studied in the context of murine sepsis. Upon cecal ligation and puncture (CLP), NF-kappaB DNA binding activity in thymocytes declines relative to sham-operated mice. This decline in NF-kappaB activity is most likely due to posttranslational modifications such as deacetylation of p65. In parallel, cleavage of procaspase-3 is increased, whereas expression of NF-kappaB-dependent antiapoptotic genes Bcl-xL and c-IAP2 is suppressed upon sepsis induction. Interestingly, adoptive transfer of IkappaBalpha-deficient fetal liver stem cells into sublethally irradiated lymphopenic host mice reduced the decline in thymocyte survival, increased peripheral T cell numbers, and improved the mortality rate relative to wild-type reconstituted hosts after cecal ligation and puncture. In conclusion, lymphocyte-directed augmentation of canonical NF-kappaB ameliorates immunosuppression during murine sepsis. These data provide evidence for a new approach in sepsis therapy.     

PI-3K/Akt and NF-kappaB/IkappaBalpha pathways are activated in Jurkat T cells in response to TRAIL treatment

The aim of this work was to evaluate the involvement of survival pathways in the response of Jurkat T leukaemic cells sensitive to the cytotoxic action of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/Apo2L. Jurkat T cells express TRAIL-R2/DR5 and TRAIL-R4/DcR2 receptors and start to die by apoptosis early (3 h) upon TRAIL administration reaching a dose-dependent increase in the percentage of dead cells within 48 h (up to 85-90%). This increase in cell death is accompanied by a dose-dependent significant (P < 0.05) increase in the G0/G1 phase of the cell cycle and reverted by the treatment with a broad inhibitor of caspases, z-VAD-fmk. Co-treatment of the cells with inhibitors of PI-3 kinase (LY294002) and nuclear factor kappa B (NF-kappaB) (SN50) pathways leads to an earlier significantly increased cytotoxicity, respectively in the form of apoptosis and necrosis. Consistently with the data obtained with the pharmacological inhibitors, the activation and nuclear translocation of both PI-3K and NF-kappaB were observed. In summary, our results provide evidence that even in sensitive neoplastic cells TRAIL paradoxically activates pro-survival pathways, which protect against TRAIL-mediated death since their inhibition leads to an earlier and increased cytotoxicity.     

NF-kappaB activation and IkappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells

This review will discuss the recent literature on the molecular mechanism of NF-kappaB activation, with special focus on IkappaB alpha dynamism involved in iNOS- and chemokine-induction in glial cells. NF-kappaB, a heterotrimer composed of p50, p65 (Rel A) and IkappaB alpha, has been shown to be activated by elimination of the regulatory subunit IkappaB alpha from the heterotrimer. The elimination of IkappaB alpha (formation of active NF-kappaB, p50-p65) is due to phosplorylation of serines 32 and 36 of IkappaB alpha, followed by polyubiquitination and 26S proteasomal degradation of IkappaB alpha. Experiments using stable clones of rat C6 glioma cells transfected with dominant negative IkappaB alpha (serines 32 and 36 replaced by alanine) suggest that NF-kappaB activation (phosphorylation of IkappaB alpha) is involved in LPS/IFNgamma- or IL-1beta/IFNgamma-induced iNOS expression. Furthermore, the time courses of phosphorylation, ubiquitination of IkappaB alpha and proteasome activity after IL-1beta treatment also suggest that 26S proteasomal degradation of IkappaB alpha is more crucial for chemokine expression in glial cells.