CD40-mediated activation of NF-kappa B in airway epithelial cells

We have reported previously that airway epithelial cells (AEC) express CD40 and that activation of this molecule stimulates the expression of inflammatory mediators, including the chemokine RANTES (regulated on activation normal T cell expressed and secreted). Because NF-kappaB regulates the expression of many inflammatory mediators, such as RANTES, we utilized CD40-mediated induction of RANTES expression to investigate the mechanisms that underlie CD40-mediated activation of NF-kappaB in AEC. Results demonstrate that, in AEC, intact NF-kappaB sites were required for CD40-mediated activation of the RANTES promoter. To examine activation of NF-kappaB binding directly, electrophoretic mobility shift analyses were performed. These analyses revealed that CD40 ligation stimulated NF-kappaB binding and that the activated NF-kappaB complexes were composed of p65 subunits. Additional studies focused on the CD40-triggered signaling pathways that facilitate NF-kappaB activation. Findings show that CD40 engagement activated the IkappaB kinases IKK-alpha and IKK-beta and stimulated IkappaBalpha phosphorylation. Analyses also examined the role of tumor necrosis factor-associated factor (TRAF) molecules in CD40-mediated NF-kappaB activation within AEC. Stable transfectants expressing wild-type or mutant forms of the cytoplasmic domain of CD40 suggested that TRAF3, but not TRAF2, binding was essential for CD40-mediated RANTES expression. Further studies indicated that exogenous expression of wild-type TRAF3 enhanced activation of the RANTES promoter, whereas exogenous expression of wild-type TRAF2 inhibited this activation; TRAF3-mediated enhancement was dependent upon NF-kappaB. Together, these findings suggest that, in AEC, ligation of CD40 regulates the expression of inflammatory mediators, such as RANTES, via activation of NF-kappaB. Moreover, these results suggest that CD40-mediated signaling in AEC differs with previously reported findings observed in other cell models, such as B lymphocytes.     

CD27 and CD40 inhibit p53-independent mitochondrial pathways in apoptosis of B cells induced by B cell receptor ligation

B cells in the germinal center are known to undergo apoptosis after B cell receptor (BCR) ligation, a process relevant to immunological tolerance. Human CD27 is a B cell co-stimulatory molecule. The aim of this study was to compare the effects of CD27 and CD40 signals on BCR-mediated apoptosis of B cells. BCR ligation activated mitochondrial apoptotic pathways including down-regulation of Bcl-X(L), dissipation of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-9. Each of these effects was significantly inhibited by CD27 and CD40. Bik expression was weakly but significantly down-regulated by CD27 but up-regulated by CD40. BCR ligation resulted in p53 activation including its phosphorylation at Ser(15), nuclear translocation, and target gene p53AIP1 induction. CD27 and CD40 clearly suppressed these processes. Analyses that used dominant-negative p53 variants revealed a low but still substantial level of BCR-mediated apoptosis and intact mitochondria-mediated apoptotic pathway. These pathways were further inhibited by CD27 and CD40, although the cells showed no p53 phosphorylation or p53AIP1 expression. Our results suggested that, at the mitochondrial level, CD27 and CD40 co-stimulatory signals regulated the p53-amplified apoptotic pathway in B cells through the inhibition of p53-independent apoptotic pathway primarily induced by BCR ligation.     

A novel mechanism for TNFR-associated factor 6-dependent CD40 signaling

Members of the TNFR family play critical roles in the regulation of the immune system. One member of the family critical for efficient activation of T-dependent humoral immune responses is CD40, a cell surface protein expressed by B cells and other APC. The cytoplasmic domain of CD40 interacts with several members of the TNFR-associated factor (TRAF) family, which link CD40 to intracellular signaling pathways. TRAF2 and 6 appear to play particularly important roles in CD40 signaling. Previous studies suggest that the two molecules have certain overlapping roles in signaling, but that unique roles for each molecule also exist. To better define the roles of TRAF2 and TRAF6 in CD40 signaling, we used somatic cell gene targeting to generate TRAF-deficient mouse B cell lines. A20.2J cells deficient in TRAF6 exhibit marked defects in CD40-mediated JNK activation and the up-regulation of CD80. Our previous experiments with TRAF2-deficient B cell lines suggest that TRAF6 and TRAF2 may have redundant roles in CD40-mediated NF-kappaB activation. Consistent with this hypothesis, we found CD40-mediated activation of NF-kappaB intact in TRAF6-deficient cells and defective in cells lacking both TRAF2 and TRAF6. Interestingly, we found that TRAF6 mutants defective in CD40 binding were able to restore CD40-mediated JNK activation and CD80 up-regulation in TRAF6-deficient cells, indicating that TRAF6 may be able to contribute to certain CD40 signals without directly binding CD40.     

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.     

OX40-mediated memory T cell generation is TNF receptor-associated factor 2 dependent

Tumor necrosis factor receptor-associated factor 2 (TRAF2), an adapter protein that associates with the cytoplasmic tail of OX40, may play a critical role in OX40-mediated signal transduction. To investigate the in vivo role of TRAF2 in OX40-mediated generation of Ag-specific memory T cells, we bred OVA-specific TCR transgenic mice to TRAF2 dominant-negative (TRAF2 DN) mice. Following Ag stimulation and OX40 engagement of TRAF2 DN T cells in vivo, the number of long-lived OVA-specific T cells and effector T cell function was dramatically reduced when compared with wild-type T cells. We also demonstrate that CTLA-4 is down-regulated following OX40 engagement in vivo and the OX40-specific TRAF2 DN defect was partially overcome by CTLA-4 blockade in vivo. The data provide evidence that TRAF2 is linked to OX40-mediated memory T cell expansion and survival, and point to the down-regulation of CTLA-4 as a possible control element to enhance early T cell expansion through OX40 signaling.     

Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.     

Tumor necrosis factor receptor-associated factor 2 (TRAF2)-deficient B lymphocytes reveal novel roles for TRAF2 in CD40 signaling

CD40 function is initiated by tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins, which play important roles in signaling by numerous receptors. Characterizing roles of individual TRAFs has been hampered by limitations of available experimental models and the poor viability of most TRAF-deficient mice. Here, B cell lines made deficient in TRAF2 using a novel homologous recombination system reveal new roles for TRAF2. We demonstrate that TRAF2 participates in synergy between CD40 and B cell antigen receptor signals, and in CD40-mediated, TNF-dependent IgM production. We also find that TRAF2 participates in the degradation of TRAF3 associated with CD40 signaling, a role that may limit inhibitory actions of TRAF3. Finally, we show that TRAF2 and TRAF6 have overlapping functions in CD40-mediated NF-kappaB activation and CD80 up-regulation. These findings demonstrate previously unappreciated roles for TRAF2 in signaling by TNF receptor family members, using an approach that facilitates the analysis of genes critical to the viability of whole organisms.     

The adaptor protein HSH2 attenuates apoptosis in response to ligation of the B cell antigen receptor complex on the B lymphoma cell line, WEHI-231

Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability.     

ER stress is involved in B cell antigen receptor ligation-induced apoptosis

Apoptosis of B cells upon ligation of the B cell antigen receptor (BCR) plays a role in elimination of self-reactive B cells. Previously, BCR ligation was shown to induce expression of the molecules involved in the unfolded protein response (UPR). However, the role of the UPR in BCR-mediated apoptosis is poorly understood. Here, we demonstrate that activation of various UPR molecules are induced when BCR ligation induces apoptosis in the B cell line WEHI-231 and mouse spleen B cells. BCR ligation-induced UPR is attenuated by survival signaling through CD40 in these cells. When overexpression of BiP suppresses the UPR in WEHI-231 cells, activation of p38 MAPK is blocked and apoptosis is reduced. Moreover, the p38 MAPK inhibitor SB203580 reduces BCR ligation-induced apoptosis. These results suggest that the UPR is involved in BCR ligation-induced apoptosis and that p38 MAPK is crucial for apoptosis during the UPR in B cells.     

Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.     

Cooperation between TNF receptor-associated factors 1 and 2 in CD40 signaling

TNFR-associated factor 1 (TRAF1) is unique among the TRAF family, lacking most zinc-binding features, and showing marked up-regulation following activation signals. However, the biological roles that TRAF1 plays in immune cell signaling have been elusive, with many reports assigning contradictory roles to TRAF1. The overlapping binding site for TRAFs 1, 2, and 3 on many TNFR superfamily molecules, together with the early lethality of mice deficient in TRAFs 2 and 3, has complicated the quest for a clear understanding of the functions of TRAF1. Using a new method for gene targeting by homologous recombination in somatic cells, we produced and studied signaling by CD40 and its viral oncogenic mimic, latent membrane protein 1 (LMP1) in mouse B cell lines lacking TRAF1, TRAF2, or both TRAFs. Results indicate that TRAFs 1 and 2 cooperate in CD40-mediated activation of the B cell lines, with a dual deficiency leading to a markedly greater loss of function than that of either TRAF alone. In the absence of TRAF1, an increased amount of TRAF2 was recruited to lipid rafts, and subsequently, more robust degradation of TRAF2 and TRAF3 was induced in response to CD40 signaling. In contrast, LMP1 did not require either TRAFs 1 or 2 to induce activation. Taken together, our findings indicate that TRAF1 and TRAF2 cooperate in CD40 but not LMP1 signaling and suggest that cellular levels of TRAF1 may play an important role in modulating the degradation of TRAF2 and TRAF3 in response to signals from the TNFR superfamily.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeostasis of the colon to prevent spontaneous development of murine inflammatory bowel disease

Fine-tuning of host cell responses to commensal bacteria plays a crucial role in maintaining homeostasis of the gut. Here, we show that tumor necrosis factor receptor-associated factor (Traf)2(-/-) mice spontaneously developed severe colitis and succumbed within 3 weeks after birth. Histological analysis revealed that apoptosis of colonic epithelial cells was enhanced, and B cells diffusely infiltrated into the submucosal layer of the colon of Traf2(-/-) mice. Expression of proinflammatory cytokines, including Tnfa, Il17a, and Ifng, was up-regulated, whereas expression of antimicrobial peptides was down-regulated in the colon of Traf2(-/-) mice. Moreover, a number of IL-17-producing helper T cells were increased in the colonic lamina propria of Traf2(-/-) mice. These cellular alterations resulted in drastic changes in the colonic microbiota of Traf2(-/-) mice compared with Traf2(+/+) mice. Treatment of Traf2(-/-) mice with antibiotics ameliorated colitis along with down-regulation of proinflammatory cytokines and prolonged survival, suggesting that the altered colonic microbiota might contribute to exacerbation of colitis. Finally, deletion of Tnfr1, but not Il17a, dramatically ameliorated colitis in Traf2(-/-) mice by preventing apoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, and restoration of wild-type commensal bacteria. Together, TRAF2 plays a crucial role in controlling homeostasis of the colon.     

Cutting edge: profound defect in T cell responses in TNF receptor-associated factor 2 dominant negative mice

TNFR-associated factor 2 (TRAF2) is an adapter protein that links several members of the TNFR family to downstream signaling pathways. Mice expressing a dominant negative form of TRAF2 in their lymphoid cells (TRAF2.DN mice) have a profound defect in T cell responses to allogeneic APC. In contrast, APC from wild-type or TRAF2.DN mice show an equivalent level of stimulation in a MLR. Ab production and class switch are unimpaired in TRAF2.DN mice. Thus, defects in the TRAF.DN mice appear to be limited to T cells. TRAF2.DN mice demonstrate an impaired T cell response to influenza virus, including decreased secondary expansion of IFN-gamma-secreting T cells as well as a decrease in CTL activity. CD4 T cell production of IL-2 was also dramatically impaired in TRAF2.DN mice. These studies suggest an essential role of TRAF2-linked receptors in secondary CD4 and CD8 T cell responses and have important implications for transplantation.     

TNFR-associated factor family protein expression in normal tissues and lymphoid malignancies

TNFR-associated factors (TRAFs) constitute a family of adapter proteins that associate with particular TNF family receptors. Humans and mice contain six TRAF genes, but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, and TRAF6. TRAF1 was also highly inducible by CD40 ligand in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immunoblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n = 49) compared with normal peripheral blood B cells (p = 0.01), as determined by immunoblotting. These findings contribute to an improved understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies.     

Cutting edge: CD40 engagement eliminates the need for Bruton's tyrosine kinase in B cell receptor signaling for NF-kappa B

The Tec kinase Bruton's tyrosine kinase (Btk) represents a key intermediary for B cell receptor (BCR) signaling. Btk mutation produces B cell deficiency in mice with X-linked immunodeficiency (xid), and surface Ig-mediated responses of mature B cells are seriously deranged. The central role that Btk plays in directing downstream events produced by BCR engagement is demonstrated by the complete failure of NF-kappa B induction and cellular proliferation following anti-Ig treatment of B cells obtained from xid mice. In this study, we report that the block in BCR signaling produced by Btk mutation is reversed by CD40 engagement. Prior treatment with CD40 ligand normalized subsequent responses of xid B cells to BCR cross-linking, so that typical outcomes of BCR signaling such as NF-kappa B activation and cell cycle progression occurred in a Btk-independent fashion. These results demonstrate that a specific genetic lesion interrupting BCR-mediated intracellular signaling is circumvented through stimulation of CD40.     

The murine NK receptor 2B4 (CD244) exhibits inhibitory function independent of signaling lymphocytic activation molecule-associated protein expression

2B4 (CD244) is a receptor belonging to the CD2-signaling lymphocytic activation molecule family and is found on all murine NK cells and a subset of NKT and CD8+ T cells. Murine 2B4 is expressed as two isoforms (2B4 short and 2B4 long) that arise by alternative splicing. They differ only in their cytoplasmic domains and exhibit opposing function when expressed in the RNK-16 cell line. The ligand for 2B4, CD48, is expressed on all hemopoietic cells. Previous studies have shown that treatment of NK cells with a 2B4 mAb results in increased cytotoxicity and IFN-gamma production. In this report, we used CD48+/- variants of the P815 tumor cell line and 2B4 knockout mice to show that engagement of 2B4 by its counterreceptor, CD48, expressed on target cells leads to an inhibition in NK cytotoxicity. The addition of 2B4 or CD48 mAb relieves this inhibition resulting in enhanced target cell lysis. This 2B4-mediated inhibition acts independently of signaling lymphocytic activation molecule-associated protein expression. Imaging studies show that 2B4 preferentially accumulates at the interface between NK and target cells during nonlytic events also indicative of an inhibitory receptor. This predominant inhibitory function of murine 2B4 correlates with increased 2B4 long isoform level expression over 2B4 short.     

B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.