TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling.     

Enhancement of MHC class I-stimulated alloresponses by TNF/TNF receptor (TNFR)1 interactions and of MHC class II-stimulated alloresponses by TNF/TNFR2 interactions

In vivo TNF inhibition has been observed to ameliorate the disease process attributed to T cell-dependent immune responses such as those generated during graft-vs.-host disease. The present studies were designed to evaluate whether TNF/TNF receptor (TNFR)1 and TNF/TNFR2 interactions were involved in the generation of allospecific T cell responses. Splenic lymphocyte populations were obtained from TNFR1- or TNFR2-deficient B6 mice and from control B6 mice. These responder cells were cultured with irradiated MHC class II-disparate B6.C-H-2bm12 (bm12) or MHC class I-disparate B6.C-H-2bm1 (bm1) or irradiated syngeneic stimulator cells for 3 days before assay of [3H]thymidine incorporation. IL-2 levels of the mixed lymphocyte culture (MLC) supernatants were assessed by enzyme-linked immunosorbent assay. With MHC class II-disparate bm12 stimulator cells, a significant reduction in T cell proliferation was observed utilizing TNFR2-deficient CD4+ responder T cells, but not when using TNFR1 -deficient CD4+ responder T cells. A significant decrease in proliferation of TNFR1-deficient CD8+ responder cells, but not of TNFR2-deficient CD8 responder T cells was observed after stimulation with MHC class I-disparate bm1 stimulator cells. IL-2 levels were lower in MLC utilizing MHC class I stimulators and TNFR1-deficient responders or MHC class II stimulators and TNFR2-deficient responders. These results indicate that TNF/TNFR2 interactions promote MHC class II-stimulated alloresponses, while TNF/TNFR1 interactions promote MHC class I-stimulated alloresponses.     

TRAF1 regulates recruitment of lymphocytes and, to a lesser extent, neutrophils, myeloid dendritic cells and monocytes to the lung airways following lipopolysaccharide inhalation

Inhaled lipopolysaccharide (LPS) induces an inflammatory response that may contribute to the pathogenesis of asthma and other airway diseases. Here we investigate the role of tumour necrosis factor (TNF) receptor-associated factor 1 (TRAF1) in leucocyte recruitment using a model of LPS-induced lung inflammation in mice. TRAF1(-/-) mice are completely deficient in the recruitment of lymphocytes to the lower respiratory tract after inhalation of LPS. Although TRAF1(-/-) mice display normal early accumulation of neutrophils, dendritic cells and monocytes in the alveolar airspace, they have a significantly reduced recruitment of these cells by 24 hr after inhalation of LPS when compared to wild-type (WT) mice. Despite normal expression of the pro-inflammatory cytokines TNF, interleukin-1 (IL-1) and IL-6 after LPS treatment, TRAF1(-/-) mice displayed decreased expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1, CCL17 and CCL20 in the lungs, when compared to LPS-treated WT mice. These results suggest that TRAF1 facilitates LPS-induced leucocyte recruitment into the lung airways by augmenting the expression of chemokines and adhesion molecules. Mice lacking TNF receptor 1 (TNFR1) but not TNFR2 show a phenotype similar to the TRAF1(-/-) mice, suggesting that TRAF1 may act downstream of TNFR1. Significantly, we use bone marrow chimeras to demonstrate that expression of TRAF1 by cells resident in the lungs, but not by circulating leucocytes, is necessary for efficient LPS-induced recruitment of leucocytes to the lung airways.     

TRAF6 negatively regulates the Jak1-Erk pathway in interleukin-2 signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a critical role in establishing both innate and acquired immune responses by mediating signals from the TNF superfamily, the TLR/IL-1R family, and the T-cell receptor. Here, we report a previously unidentified function of TRAF6 in IL-2 signaling. CD3/CD28 stimulation-induced proliferation and Il2 mRNA expression in Traf6(-/-) CD4(+) T cells were dramatically enhanced. This enhancement is likely due to hyperactive IL-2 signaling, in which activation of the Jak1-Erk pathway was enhanced and the subsequent Fos gene expression was up-regulated. To elucidate the molecular mechanisms of the enhanced activation of Jak1, IL-2 signaling was reconstituted in mouse embryonic fibroblast (MEF) cells to investigate the interaction between TRAF6 and the TRAF6-binding site that overlaps with the Jak1-binding site present in the IL-2R β-chain. The Jak1-Erk pathway was activated upon IL-2 stimulation in Traf6(-/-) MEF cells, while a β-chain mutation that inactivates TRAF6 binding but retains Jak1 binding abrogated the TRAF6-dependent reduction in IL-2 signaling. These results indicate that the binding of TRAF6 to the TRAF6-binding site of the β-chain negatively regulates IL-2-induced Jak1 activation, which is likely to be involved in the proper regulation of T-cell activation and development.     

Collagen-induced arthritis in TNF receptor-1-deficient mice: TNF receptor-2 can modulate arthritis in the absence of TNF receptor-1

TNF is a potent proinflammatory cytokine important for the development of arthritis in human and animals. We have investigated the roles of TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in collagen-induced arthritis (CIA) by inducing CIA in mice genetically deficient in TNFR1. TNFR1-/- mice developed arthritis with similar incidence and severity as TNFR1+/- littermates, indicating that TNFR1 is redundant for the development of CIA. Anti-type II collagen (CII) antibody levels and T cell responses to CII did not differ between TNFR1-/- mice and controls. Neutralization of TNF with soluble TNF binding protein suppressed the development of arthritis in TNFR1+/- mice but not in TNFR1-/- mice, indicating that TNFR2 cannot substitute for TNFR1 for the proinflammatory function. To further investigate the functions of TNFR2, TNFR1-/- mice were injected with murine TNF-alpha at different stages during the course of CIA. Repeated TNF-alpha injection during the early induction phase enhanced the development of arthritis, but inhibited arthritis when administered during the late progression phase. These results show that the engagement of TNFR2 by TNF is involved in the development of CIA in the absence of TNFR1 and that opposing signals can be transduced by TNFR2.     

TNFR1 delivers pro-survival signals that are required for limiting TNFR2-dependent activation-induced cell death (AICD) in CD8+ T cells

Members of the TNF and TNF receptor (TNFR) superfamily play important roles in the maintenance of homeostasis of the immune system. Furthermore, several members of the TNFR family participate in T-cell activation and sustaining T-cell responses. We have shown that TNFR2 regulates T-cell activation by lowering the activation threshold and providing costimulatory signaling. Furthermore, activated TNFR2(-/-) CD8(+) T cells are highly resistant to activation-induced cell death (AICD). Here, we showed that using anti-TNFR2 antibodies to block TNFR2 on activated WT CD8(+) T cells rendered them resistant to AICD. This resistance of activated TNFR2(-/-) CD8(+) T cells to AICD correlated with the accumulation of TNF receptor-associated factor 2 (TRAF2). Overexpression of TRAF2 by retroviral transfection and knockdown of TRAF2 by small interfering RNA also support this conclusion. Furthermore, neutralizing TNF-α reduced TRAF2 accumulation in activated TNFR2(-/-) CD8(+) T cells and increased their susceptibility to AICD. AICD-resistant TNFR2(-/-) CD8(+) T cells expressed elevated levels of phosphorylated IκBα and higher DNA-binding activity of the p65 NK-κB subunit and neutralization of TNF-α blocked this increase. Therefore, in activated TNFR2(-/-) CD8(+) T cells, TNFR1 functions as a survival receptor by utilizing high intracellular levels of TRAF2 to promote IκBα phosphorylation and NF-κB activation.     

TRAF6 is a critical factor for dendritic cell maturation and development

IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily members are critical for regulating multiple aspects of dendritic cell (DC) biology. Several signaling pathways associated with each family utilize the adapter molecule, TRAF6, but its role in DCs is unclear. By examining TRAF6-deficient mice and bone marrow (BM) chimeras reconstituted with TRAF6-deficient fetal liver cells, we show that proper DC maturation requires TRAF6. In response to either microbial components or CD40L, TRAF6-deficient DCs fail to upregulate surface expression of MHCII and B7.2, or produce inflammatory cytokines. Moreover, LPS-treated TRAF6-deficient DCs do not exhibit an enhanced capacity to stimulate naive T cells. Interestingly, a major population of splenic DCs, the CD4(+)CD8alpha(-) subset, is nearly absent in both TRAF6-deficient mice and BM chimeras. Together these results indicate that TRAF6 regulates the critical processes required for maturation, activation, and development of DCs, the primary cellular bridge between innate and adaptive immunity.     

Acquisition of regulatory function by human CD8(+) T cells treated with anti-CD3 antibody requires TNF

Anti-CD3 mAb can modulate graft rejection and attenuate autoimmune diseases but their mechanism(s) of action remain unclear. CD8(+) T cells with regulatory function are induced in vitro by Teplizumab, a humanized anti-CD3 antibody and inhibit responses of autologous and allogeneic T cells. They inhibit CD4(+) T-cell proliferation by mechanisms involving TNF and CCL4, and by blocking target cell entry into G2/M phase of cell cycle but neither kill them, nor compete for IL-2. CD8(+) Treg can be isolated from peripheral blood following treatment of patients with Type 1 diabetes with Teplizumab, but not from untreated patients. The induction of CD8(+) Treg by anti-CD3 mAb requires TNF and signaling through the NF-κB cascade. The CD8(+) Treg express CD25, glucocorticoid-induced TNF receptor family, CTLA-4, Foxp3, and TNFR2, and the combined expression of TNFR2 and CD25 identifies a potent subpopulation of CD8(+) Treg. These studies have identified a novel mechanism of immune regulation by anti-CD3 mAb and markers that may be used to track inducible CD8(+) Treg in settings such as chronic inflammation or immune therapy.     

Tumor necrosis factor-alpha-induced lung cell expression of antiapoptotic genes TRAF1 and cIAP2

Tumor necrosis factor (TNF) receptor (TNFR)-associated factors 1 and 2 (TRAF1 and TRAF2) and inhibitor of apoptosis proteins cIAP1 (MIHB) and cIAP2 (MIHC) were recently identified as proteins that associate with the TNF-alpha receptors TNFRI (p55) and TNFRII (p75) and inhibit TNF-alpha-induced programmed cell death or apoptosis. In the original reports, TRAF1 expression, unlike the ubiquitous TRAF2, was restricted to specific tissues in the lung, spleen, and testis. TNF-alpha is increased in the lung in many forms of pulmonary disease. In the current study, Western analysis, immunohistochemistry, and ribonuclease protection assays were used to determine whether TNF-alpha regulates the expression of these TNFR-associated proteins in lung cells. We demonstrate for the first time TNF-alpha dose-dependent induction of TRAF1 protein and messenger RNA (mRNA) in human H441 and A549 pulmonary adenocarcinoma cell lines, as well as in lung cells of C57BL/6J mice after intratracheal administration of TNF-alpha. In contrast to the epithelial cells, TRAF1 was not induced by TNF-alpha in U937 cells, a human monocytic cell line, suggesting cell type-specific regulation. Similarly, cIAP2 mRNA was induced by TNF-alpha in both H441 and A549 pulmonary epithelial cells but not in U937 cells. TNF-alpha is a primary mediator of acute pulmonary inflammation and contributes to the pathophysiology of chronic lung diseases such as bronchopulmonary dysplasia (BPD), a fibrotic disease of prematurely born infants. Immunohistochemical staining of human neonatal lung tissue demonstrated increased TRAF1 in lungs of infants dying of pneumonia or BPD in comparison with those dying of congenital malformation. These studies support the hypothesis that the TRAF1 and cIAP2 genes are highly regulated in pulmonary cells and may play a role in human lung disease.     

TNF receptor-associated factor-1 (TRAF1) negatively regulates Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-mediated signaling

Toll-like receptor 3 (TLR3) plays an important role in antiviral responses through recognizing viral double-stranded RNA produced during viral infection and mediating induction of type I IFN. TRIF is a Toll/IL-1 receptor (TIR) domain-containing adaptor protein that is associated with TLR3 and critically involved in TLR3-mediated signaling. In yeast two-hybrid screens, we identified TNF receptor-associated factor (TRAF)1 as a TRIF-interacting protein. The TRAF-C domain of TRAF1 and the TIR domain of TRIF were responsible for their interaction. Overexpression of TRAF1 inhibited TRIF- and TLR3-mediated activation of NF-kappaB, IFN-stimulated response element and the IFN-beta promoter. Overexpression of TRIF caused caspase-dependent cleavage of TRAF1. The cleaved N-terminal but not C-terminal fragment of TRAF1 was responsible for inhibiting TRIF signaling. Mutation of the caspase cleavage site of TRAF1 or addition of the caspase inhibitor crmA inhibited TRAF1 cleavage and abolished the ability of TRAF1 to inhibit TRIF signaling, suggesting that TRIF-induced cleavage of TRAF1 is required for its inhibition of TRIF signaling. Our findings provide a novel mechanism for negative regulation of TRIF-mediated signaling.     

TNF optimally activatives regulatory T cells by inducing TNF receptor superfamily members TNFR2, 4-1BB and OX40

TNF is a pleiotropic cytokine with intriguing biphasic pro-inflammatory and anti-inflammatory effects. Our previous studies demonstrated that TNF up-regulated FoxP3 expression and activated and expanded CD4+ FoxP3+ regulatory T cells (Tregs) via TNFR2. Furthermore, TNFR2-expressing Tregs exhibited maximal suppressive activity. In this study, we show that TNF, in concert with IL-2, preferentially up-regulated mRNA and surface expression of TNFR2, 4-1BB and OX40 on Tregs. Agonistic antibodies against 4-1BB and OX40 also induced the proliferation of suppressive Tregs. Thus, TNF amplifies its stimulatory effect on Tregs by inducing TNF receptor superfamily (TNFRSF) members. In addition, administration of neutralizing anti-TNF Ab blocked LPS-induced expansion of splenic Tregs and up-regulation of TNFR2, OX40 and 4-1BB receptors on Tregs in vivo, indicating that the expansion of Tregs expressing these co-stimulatory TNFRSF members in response to LPS is mediated by TNF. Altogether, our novel data indicate that TNF preferentially up-regulates TNFR2 on Tregs, and this is amplified by the stimulation of 4-1BB and OX40, resulting in the optimal activation of Tregs and augmented attenuation of excessive inflammatory responses.     

TNF-induced enterocyte apoptosis in mice is mediated by the TNF receptor 1 and does not require p53

Injection of recombinant mouse TNF into mice is known to induce a shrinkage of the duodenal villi, which becomes evident 30 – 90 min later and is associated with a detachment of enterocytes in the lumen. These cells can be collected by lavage and are all apoptotic, i.e. hypodiploid as seen by flow cytometric analysis. Thus the count of detached cells was used as an evaluation of the TNF-induced cell loss and apoptosis in the mucosa. TNF injection induced a cell loss of similar magnitude in wild-type (+/+) or in mice lacking the TNF receptor (TNFR)2 (p75, TNFR2 −/−), while mice lacking the TNFR1 (p55, TNFR1 −/−) were completely resistant to this effect. TNF increased the expression of p53 tumor suppressor gene in the enterocytes from the crypts but not from the villi, as seen by Western blots and histochemistry. TNF increased the expression of p53 in both TNFR2 −/− and TNFR1 −/− mice. Furthermore, enterocyte cell loss was not attenuated in p53 −/− mice. The results indicate that TNF, acting on its receptor 1, induces an apoptotic detachment of the enterocytes from the tip of the villi ( i.e. the old enterocytes), while in the enterocytes from the crypts (the young enterocytes) TNF increases, via either TNFR1 or TNFR2, the expression of p53, without inducing apoptosis.     

TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.     

The binding site for TRAF2 and TRAF3 but not for TRAF6 is essential for CD40-mediated immunoglobulin class switching

To define the role of TRAF proteins in CD40-dependent isotype switching in B cells, we introduced wild-type (WT) and mutant CD40 transgenes that lacked the binding motifs for TRAF6 (CD40deltaTRAF6), TRAF2 and TRAF3 (CD40deltaTRAF2/3), or both (CD40deltaTRAFs) into B cells of CD40(-/-) mice. The in vivo isotype switch defect in CD40(-/-) mice was fully corrected by WT and CD40deltaTRAF6, partially by CD40deltaTRAF2/3, and not at all by CD40deltaTRAFs transgenes. CD40-mediated isotype switching, proliferation, and activation of p38, JNK, and NFkappaB in B cells were normal in WT and CD40deltaTRAF6 mice, severely impaired in CD40deltaTRAF2/3, and absent in CD40deltaTRAFs mice. These results suggest that binding to TRAF2 and/or TRAF3 but not TRAF6 is essential for CD40 isotype switching and activation in B cells.     

TNF and TNF receptor polymorphisms in Korean Behcet's disease patients

Behcet's disease (BD) is an autoimmune disease characterized by recurrent oral ulcers, genital ulcers, erythema nodosum, and uveitis. Genetic factors are considered important in its pathogenesis. The serum level of tumor necrosis factor (TNF) is elevated in patients with active BD, and its production is elevated in monocytes and in the gamma delta T cells of BD patients. A dramatic response to anti-TNF-alpha antibody treatment further supports the role of TNF in BD. In this study, we investigated genetic polymorphisms of TNF alpha -308 G/A, TNF beta +252 G/A, and TNFR2 196 R/M in 94 Korean BD patients and age- and sex-matched healthy controls to investigate the role of TNF and TNF receptor polymorphisms in BD. The polymerase chain reaction-restriction fragment length polymorphism was used to identify the TNF-alpha promoter (G = TNFA1, A = TNFA2) and TNF-beta intron polymorphisms (G = TNFB1, A = TNFB2), and polymerase chain reaction-singly-strand conformation polymorphism was used to identify TNFR2 196R/M polymorphism (T = TNFR2M, G = TNFR2R). No differences were found in the TNF-alpha, TNF-beta or TNFR2 polymorphisms of the patients and the healthy controls. The allele frequencies of TNFA1/A2 were 0.94/0.06 in patients and 0.96/0.04 in healthy controls (p = 0.36, OR = 0.65, 95% CI = 0.26-1.63), for TNFB1/TNFB2 these were 0.42/0.58 in patients and 0.44/0.56 in controls (p = 0.68, OR = 0.91, 95% CI = 0.61-1.38), and for TNFR2R/TNFR2M 0.23/0.77 in patients and 0.21/0.79 in controls (p = 0.62, OR = 1.13, 95% CI = 0.69-1.84). In conclusion, this study found no differences of TNF alpha -308 G/A, TNF beta +252 G/A or of the TNFR2 196R/M polymorphisms in Korean BD patients versus healthy controls. These findings suggest that the role of TNF in BD is not genetically determined, but can be functionally explained.     

TRAF2 regulates T cell immunity by maintaining a Tpl2-ERK survival signaling axis in effector and memory CD8 T cells

Generation and maintenance of antigen-specific effector and memory T cells are central events in immune responses against infections. We show that TNF receptor-associated factor 2 (TRAF2) maintains a survival signaling axis in effector and memory CD8 T cells required for immune responses against infections. This signaling axis involves activation of Tpl2 and its downstream kinase ERK by NF-κB-inducing kinase (NIK) and degradation of the proapoptotic factor Bim. NIK mediates Tpl2 activation by stimulating the phosphorylation and degradation of the Tpl2 inhibitor p105. Interestingly, while NIK is required for Tpl2-ERK signaling under normal conditions, uncontrolled NIK activation due to loss of its negative regulator, TRAF2, causes constitutive degradation of p105 and Tpl2, leading to severe defects in ERK activation and effector/memory CD8 T cell survival. Thus, TRAF2 controls a previously unappreciated signaling axis mediating effector/memory CD8 T cell survival and protective immunity.     

Differential functions of tumor necrosis factor receptor 1 and 2 signaling in ischemia-mediated arteriogenesis and angiogenesis

We have previously shown that tumor necrosis factor (TNF) acts via its two receptors TNFR1 and TNFR2 to elicit distinct signaling pathways in vascular endothelial cells (ECs). Here we used a femoral artery ligation model to demonstrate that TNFR1-knockout (KO) mice had enhanced, whereas TNFR2-KO had reduced, capacity in clinical recovery, limb perfusion, and ischemic reserve capacity compared with the wild-type mice. Consistently, ischemia-initiated collateral growth (arteriogenesis) in the upper limb and capillary formation and vessel maturation (angiogenesis) in the lower limb were enhanced in TNFR1-KO but were reduced in TNFR2-KO mice. Furthermore, our results suggest that vascular proliferation, but not infiltration of macrophages and lymphocytes, accounted for the phenotypic differences between the TNFR1-KO and TNFR2-KO mice. In wild-type animals TNFR2 protein in vascular endothelium was highly up-regulated in response to ischemia, leading to increased TNFR2-specific signaling as determined by the formation TNFR2-TRAF2 complex and activation of TNFR2-specific kinase Bmx/Etk. In isolated murine ECs, activation of TNFR2 induced nuclear factor-kappaB-dependent reporter gene expression, EC survival, and migration. In contrast, activation of TNFR1 caused inhibition of EC migration and EC apoptosis. These data demonstrate that TNFR1 and TNFR2 play differential roles in ischemia-mediated arteriogenesis and angiogenesis, partly because of their opposite effects on EC survival and migration.