Activation of the NF-kappaB signalling pathway in diffuse large B-cell lymphoma: clinical implications

Aim: To characterize the activation of the nuclear factor (NF)‐κB pathway in diffuse large B‐cell lymphoma (DLBCL) by immunohistochemistry. Methods and results: Sixty‐six DLBCLs treated with anthracycline‐containing chemotherapy were evaluated with antibodies against phosphorylated p65 (P‐p65), p65, p50, p52, IKKα, and phosphorylated IκB (P‐IκB). NF‐κB activation was based on the expression of P‐p65, P‐IκB, and nuclear expression of p65 or p52 in the tumour cells. P‐p65 and P‐IκB were expressed in 13 (20%) and 17 cases (26%), respectively. p65, p52 and IKKα were found in the cytoplasm. A correlation was found between expression of P‐p65 and P‐IκB (P < 0.0001), but not between the two subtypes of DLBCL [germinal centre B cell and non‐germinal centre (GC)]. P‐p65+ tumours showed a better response to chemotherapy (P = 0.025) and a trend to increased event‐free survival (P = 0.08). However, P‐IκB expression was not associated with either clinical response or survival. Bcl‐2 was not preferentially expressed on DLBCL tumours with NF‐κB activation, as determined by expression of P‐p65 and P‐IκB proteins. Conclusions: NF‐κB activation in DLBCL is preferentially mediated through the classical pathway and a novel mechanism involving phosphorylation of p65. Activation of NF‐κB by P‐p65 is associated with good prognosis. NF‐κB activation is not confined to non‐GC DLBCL exclusively.     

NF-kappaB p50 and p65 subunits control intestinal homeostasis

Mice which lack the p50 subunit of NF-kappaB and are heterozygous for the p65 subunit (3X mice), are exquisitely sensitive to LPS-induced shock. Here, we demonstrate that prior to becoming moribund, 3X mice challenged with LPS develop a profound enteropathy. The enteropathy is characterized by defects in intestinal barrier function, increased epithelial apoptosis, and deregulated intestinal cytokine gene expression. The defect that sensitizes 3X mice to LPS-induced enteropathy is located within the innate immune compartment, as LPS induced similar findings in 3X mice lacking lymphocytes (3X/RAG). TNF-alpha depletion ameliorated the ability of LPS to induce pathology and TNF-alpha was able to independently induce similar findings, suggesting that TNF-alpha plays a critical role in the development of LPS-induced pathology in these mice. These data highlight that NF-kappaB subunits have essential functions in regulating intestinal homeostasis during acute inflammation.     

Escherichia coli up-regulates proinflammatory cytokine expression in granulocyte/macrophage lineages of CD34 stem cells via p50 homodimeric NF-kappaB

Umbilical cord blood has emerged as an alternative source of haematopoietic CD34+ cells for allogeneic stem cell transplantation. Although bacteraemia induced by Escherichia coli is considered one of the complications of transplantation, expression of proinflammatory cytokines is poorly understood. In this study, we report the altered expression of proinflammatory cytokines in CD34+ cells and their in vitro cultured cells following E. coli infection. CD34+ stem cells and their cultured cells up-regulated expression of proinflammatory cytokines such as interleukin (IL)-1alpha, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha after infection with E. coli. Expression of the proinflammatory cytokines was generated mainly by the granulocyte-macrophage lineages. E. coli infection activated the signals of p50/p50 nuclear factor-kappaB (NF-kappaB) homodimers and IkappaB kinase. Furthermore, inhibition of NF-kappaB activation lowered the up-regulated expression of the proinflammatory cytokines. These results suggest that CD34+ cells and their cultured cells infected with E. coli induce the expression of proinflammatory cytokines via the NF-kappaB pathway.     

Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-kappaB translocation

Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor. When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4+ T cell responses. In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by up-regulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses. On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT. Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-kappaB in DC in a GM1-dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.     

Essential role for platelet-activating factor-induced NF-kappaB activation in macrophage-derived angiogenesis

Activated monocyte-macrophages have been implicated in tumor angiogenesis via their capacity to produce many potent angiogenic factors. However, the mechanisms leading to production of these angiogenic factors in macrophages remain to be elucidated. In this study, we demonstrated by use of a mouse Matrigel implantation model that mouse peritoneal macrophages induce angiogenesis. mRNA expression and protein synthesis of macrophage-derived crucial angiogenic factors such as IL-1, TNF-alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) were blocked by platelet-activating factor (PAF) receptor antagonists. It was also observed that inhibitors of NF-kappaB blocked macrophage production of these angiogenic factors. Gene expression and protein synthesis of the angiogenic factors cited above were also inhibited in IkappaBalpha-mutated macrophages. VEGF is the most potent angiogenic factor in macrophage-induced angiogenesis. PAF antagonists or NF-kappaB inhibitors also inhibit the capacity of conditioned medium from LPS-stimulated human peripheral blood monocytes to induce sprouting of porcine pulmonary arterial endothelial cells. These data indicate that PAF-induced NF-kappaB activation is a common upstream pathway leading to the production of crucial macrophage-derived angiogenic factors. This will provide an important clue for a better understanding of mechanisms involved in tumor angiogenesis.     

Caspase-8 and caspase-10 activate NF-kappaB through RIP,NIK and IKKalpha kinases

NF-kappaB regulates the expression of various genes involved in cell growth and differentiation, immune response and inhibition of apoptosis. Recently, some death effector domain (DED)-containing proteins, such as FADD and c-FLIP were reported to activate NF-kappaB. We previously reported that the prodomain-only isoforms of caspase-8 and -10 (PDCasp8/10), containing two DED motifs, could inhibit Fas-mediated apoptosis. Here, we demonstrate that these isoforms also activate NF-kappaB, implying this to be one of the mechanisms by which these polypeptides inhibit apoptosis. The GST pull-down assay revealed that, among upstream kinases that activate NF-kappaB, only NIK and RIP, but not RICK or IKKalpha/beta, could directly bind to PDCasp8/10. In addition, both modules ofDED in PDCasp8/10 were required for these interactions as well as NF-kappaB activation. Experiments using a kinase-dead mutant of IKKalpha and an RIP mutant lacking a kinase domain, both of which function as dominant-negative mutants for their wild-type counterparts, blocked PDCasp8/10-mediated NF-kappaB activation. Using small interfering RNA technology, we further demonstrate that the down-regulation of IKKalpha but not IKKbeta significantly inhibits PDCasp8-mediated NF-kappaB activation. Taken together, these results suggest that caspase-8 and -10 have roles in a non- or anti-apoptotic signaling pathway leading to NF-kappaB activation through RIP, NIK and IKKalpha.     

Direct role of NF-kappaB activation in Toll-like receptor-triggered HLA-DRA expression

Microbial components, such as DNA containing immunostimulatory CpG motifs (CpG-DNA) and lipopolysaccharides (LPS), elicit the cell surface expression of MHC class II (MHC-II) through Toll-like receptor (TLR)/IL-1R. Here, we show that CpG-DNA and LPS induce expression of the HLA-DRA in the human B cell line, RPMI 8226. Ectopic expression of the dominant negative mutant of CIITA and RNA interference targeting the CIITA gene indicate that CIITA activation is not enough for the maximal MHC-II expression induced by CpG-DNA and LPS. Additionally, nuclear factor (NF)-kappaB activation is required for the CpG-DNA-activated and LPS-activated HLA-DRA expression, whereas IFN-gamma-induced MHC-II expression depends on CIITA rather than on NF-kappaB. Comprehensive mutant analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays, reveal that the functional interaction of NF-kappaB with the promoter element is necessary for the TLR-mediated HLA-DRA induction by CpG-DNA and LPS. This novel mechanism provides the regulation of MHC-II gene expression with complexity and functional diversity.     

Increased NF-kappa B activity in B cells and bone marrow-derived dendritic cells from NOD mice

Type 1 diabetes results from the breakdown of peripheral tolerance. As regulators of T cell activation, antigen-presenting cells (APC) modulate peripheral tolerance and hence contribute to the immune dysregulation characteristic of insulin-dependent diabetes mellitus (IDDM). We initially observed an increased importance of NOD B cell APC function in a T cell priming assay as compared to non-autoimmune strains. Consistent with this increased APC function, we found that NF-kappa B nuclear translocation is increased in unmanipulated NOD and NOD.B10Sn-H2(b) B cells and that, in addition, NOD B cells are more sensitive to NF-kappa B-activating stimuli. We obtained similar results using NOD bone marrow-derived dendritic cell (BMDC) cultures. As costimulatory molecules have been shown to be NF-kappa B responsive, we examined the expression of these markers on NOD APC. Both B cells and BMDC expressed elevated levels of CD80 and CD40. Finally, NOD B cells provided better allostimulation than B cells from non-autoimmune strains. Therefore, hyperactivation of NF-kappa B and increased expression of CD80 and CD40 by NOD B cells and BMDC may be a contributing factor in the selection of effector T cells observed in IDDM.     

Induction of proinflammatory mediators requires activation of the TRAF, NIK, IKK and NF-kappaB signal transduction pathway in astrocytes infected with Escherichia coli

Escherichia coli is associated with inflammation in the brain. To investigate whether astrocytes are involved in E. coil-induced inflammation, we assessed the levels of expression of proinflammatory mediators produced by E. coli-infected astrocytes. E. coli infection in primary human astrocytes and cell lines increased expression of the CXC chemokine IL-8/GRO-alpha, the CC chemokine MCP-1, TNF-alpha, and iNOS. E. coli infection activated p65/p50 heterodimeric NF-kappaB and concurrently decreased the signals of IkappaBalpha. Blocking the NF-kappaB signals by IkappaBalpha-superrepressor-containing retrovirus or antisense p50 oligonucleotide transfection resulted in down-regulation of expression of the proinflammatory mediators. Furthermore, superrepressors of IkappaBalpha, IkappaB kinase (IKK) or NF-kappaB inducing kinase (NIK) inhibited the up-regulated expression of the downstream target genes of NF-kappaB such as IL-8 and MCP-1, and superrepressors of TNF receptor-associated factor (TRAF)2 and TRAF5 also inhibited expression of the E. coli-induced target genes of NF-kappaB. These results indicate that proinflammatory mediators such as the CXC chemokine IL-8/GRO-alpha, the CC chemokine MCP-1, TNF-alpha, and iNOS can be expressed in E. coli-infected astrocytes via an NF-kappaB pathway, suggesting that these mediators may contribute to inflammation in the brain, including infiltration of inflammatory cells.