Hsiou-Chi Liou

Targeted disruption of the p50 subunit of NF-κB leads to multifocal defects in immune responses

NF-kappa B, a heterodimeric transcription factor composed of p50 and p65 subunits, can be activated in many cell types and is thought to regulate a wide variety of genes involved in immune function and development. Mice lacking the p50 subunit of NF-kappa B show no developmental abnormalities, but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection. B cells do not proliferate in response to bacterial lipopolysaccharide and are defective in basal and specific antibody production. Mice lacking p50 are unable effectively to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae, but are more resistant to infection with murine encephalomyocarditis virus. These data support the role of NF-kappa B as a vital transcription factor for both specific and nonspecific immune responses, but do not indicate a developmental role for the factor.

Modulation of NF-κB Activity and Apoptosis in Chronic Lymphocytic Leukemia B Cells

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-κB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-κB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-κB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-κB activity and prolonged CLL cell survival in vitro. The principle NF-κB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-κB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-κB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction.

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.

NF-κB1 p50 Is Required for BLyS Attenuation of Apoptosis but Dispensable for Processing of NF-κB2 p100 to p52 in Quiescent Mature B Cells

B lymphocyte stimulator (BLyS), a TNF family protein essential for peripheral B cell development, functions primarily through attenuation of B cell apoptosis. In this study, we show that BLyS activates NF-κB through both classical and alternative pathways with distinct kinetics in quiescent mature B cells. It rapidly and transiently enhances the p50/p65 DNA binding activity and induces phosphorylation of IκBα characteristic of the classical NF-κB pathway, albeit maintaining IκBα at a constant level through ongoing protein synthesis and proteasome-mediated destruction. With delayed kinetics, BLyS promotes the processing of p100 to p52 and sustained formation of p52/RelB complexes via the alternative NF-κB pathway. p50 is dispensable for p100 processing. However, it is required to mediate the initial BLyS survival signals and concomitant activation of Bcl-xL in quiescent mature B cells ex vivo. Although also a target of BLyS activation, at least one of the A1 genes, A1-a, is dispensable for the BLyS survival function. These results suggest that BLyS mediates its survival signals in metabolically restricted quiescent B cells, at least in part, through coordinated activation of both NF-κB pathways and selective downstream antiapoptotic genes.

Essential Roles of c-Rel in TLR-Induced IL-23 p19 Gene Expression in Dendritic Cells

IL-23 plays crucial roles in both immunity against pathogens and autoimmunity against self. Although it is well recognized that IL-23 expression is restricted to the myeloid lineage and is tightly regulated at the transcriptional level, the nature of transcription factors required for IL-23 expression is poorly understood. We report, in this study, that murine dendritic cells deficient in c-Rel, a member of the NF-κB family, are severely compromised in their ability to transcribe the p19 gene, one of the two genes that encode the IL-23 protein. The p19 gene promoter contains three putative NF-κB binding sites, two of which can effectively bind c-Rel as determined by chromatin immunoprecipitation and EMSA. Unexpectedly, mutation of either of these two c-Rel binding sites completely abolished the p19 promoter activity induced by five TLRs (2, 3, 4, 6, and 9) and four members of the NF-κB family (c-Rel, p65, p100, and p105). Based on these observations, we conclude that c-Rel controls IL-23 p19 gene expression through two κB sites in the p19 promoter, and propose a c-Rel-dependent enhanceosome model for p19 gene activation.

c‐Rel is essential for B lymphocyte survival and cell cycle progression

c‐Rel is a lymphoid‐specific member of the NF‐κB / Rel family of transcriptional factors. To investigate the role of c‐Rel in B lymphocyte function, we generated a c‐Rel(− / −) mouse via a gene targeting approach. Although early lymphocyte development is normal in c‐Rel(− / −) mice, there are significantly fewer B cells displaying a memory (IgM / IgD−) phenotype. Upon immunization, c‐Rel(− / −) mice generate fewer B cells with a germinal center (PNAhi) phenotype. In vitro, c‐Rel(− / −) B cells proliferate poorly upon ligation of their surface IgM or CD40 receptors or when stimulated with either lipopolysaccharide (LPS) or T cell help. Early molecular events that precede proliferation, such as increases in RNA synthesis as well as IL‐2 receptor α chain expression, are greatly diminished in c‐Rel(− / −) B cells. Furthermore, c‐Rel(− / −) B cells are impaired in the ability to receive survival signals generated by anti‐IgM or LPS. In contrast, CD40‐mediated cell survival is normal in c‐Rel(− / −) B cells, suggesting the involvement of a survival‐signaling pathway that is independent of c‐Rel. When c‐Rel (− / −) B cells are co‐stimulated with either anti‐IgM and CD40 or LPS and CD40, they are rendered capable of progressing through the cell cycle. Finally, co‐culture experiments suggest that the defects observed in c‐Rel(− / −) B cells are intrinsic to the cell and can not be rescued through either cell‐cell contact or addition of soluble factors. Thus, c‐Rel is requisite for differentiation to the germinal center and memory B cells in vivo and is required for the transduction of survival and cell cycle progression signals mediated by anti‐IgM and LPS in vitro. Furthermore, while c‐Rel is involved in CD40‐induced proliferation, it is apparently dispensable for the survival signals transduced by CD40.

The NF-kappa B p50 precursor, p105, contains an internal I kappa B-like inhibitor that preferentially inhibits p50.

The p50 subunit of NF‐kappa B is apparently synthesized as a precursor molecule of 105 kDa (p105); subsequent processing releases the amino‐terminal p50 polypeptide with rel homology, DNA binding activity and transcriptional activation potential. The carboxy‐terminal region of p105 contains seven copies of an ankyrin‐related sequence previously found in several genes involved in differentiation and cell cycle control. Two proteins with I kappa B activity, MAD‐3 and pp40, have been cloned and found to contain five obvious ankyrin repeats that align with those in the carboxy‐terminus of p105. Both proteins target their inhibitory activity to the p65 subunit of NF‐kappa B and to c‐rel. Here we show that the bacterially expressed and purified carboxy‐terminal region (CTR) of p105 abolishes the binding of p50 homodimers to a kappa B motif but minimally affects the binding of p65 homodimers and NF‐kappa B. By contrast, MAD‐3 inhibits the binding of p65 and NF‐kappa B but not p50. Both the CTR and MAD‐3 interact with their respective targets through physical association both in vitro and in vivo. The CTR can be expressed as an independent entity and thus may play two roles, as a cis inhibitor built into the p105 molecule and as a trans regulator of p50.

Cutting Edge: Identification of c-Rel-Dependent and -Independent Pathways of IL-12 Production During Infectious and Inflammatory Stimuli

The production of IL-12 is required for immunity to many intracellular pathogens. Recent studies have shown that c-Rel, a member of the NF-κB family of transcription factors, is essential for LPS-induced IL-12p40 production by macrophages. In this study, we demonstrate that c-Rel is also required for IL-12p40 production by macrophages in response to Corynebacterium parvum, CpG oligodeoxynucleotides, anti-CD40 and low molecular weight hyaluronic acid. However, c-Rel−/− mice infected with Toxoplasma gondii produce comparable amounts of IL-12p40 to infected wild-type mice and have an IL-12-dependent mechanism of resistance to this infection. Furthermore, c-Rel was not required for IL-12p40 production by macrophages or dendritic cells in response to soluble Toxoplasma Ag, and neutrophils from c-Rel−/− mice contain normal amounts of preformed IL-12p40. Together these studies reveal the presence of c-Rel-dependent pathways critical for IL-12p40 production in response to inflammatory stimuli and demonstrate a novel c-Rel-independent pathway of IL-12p40 production during toxoplasmosis.

Phosphatidylinositol 3-Kinase and NF-κB/Rel Are at the Divergence of CD40-Mediated Proliferation and Survival Pathways

CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-κB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-κB/Rel activation. This is achieved via activation of AKT and the degradation of IκBα. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-κB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-κB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-κB/Rel and PI-3K are crucial for CD40-induced proliferation.

Differential Requirement for NF-κB Family Members in Control of Helminth Infection and Intestinal Inflammation

The NF-κB family of transcription factors is critical in controlling the expression of a wide range of immune response genes. However, whether individual family members perform specific roles in regulating immunity and inflammation remains unclear. Here we investigated the requirement for NF-κB1, NF-κB2, and c-Rel in the expression of Th2 cytokine responses, development of host protective immunity, and regulation of intestinal inflammation following infection with the gut-dwelling helminth parasite Trichuris muris. While mice deficient in c-Rel mounted sufficient Th2 responses to expel infection, NF-κB1 knockout (KO) and NF-κB2 KO mice developed chronic infections associated with elevated production of Ag-specific IFN-γ. However, only infected NF-κB1 KO mice exhibited polarized IFN-γ responses associated with the loss of intestinal goblet cells and the development of destructive colitis-like pathology. Furthermore, blockade of IL-12 (previously shown to confer resistance in susceptible strains) recovered Ag-specific IL-13 responses and resistance to infection in NF-κB2 KO, but not NF-κB1 KO mice. Therefore, unique infection, immunological, and pathological outcomes were observed in different NF-κB KO strains. Taken together, these results provide direct evidence of nonoverlapping functions for NF-κB family members in the development of Th2 cytokine-mediated resistance to T. muris and the control of infection-induced intestinal inflammation.