The role of apoptosis signal-regulating kinase 1 in lymphotoxin-beta receptor-mediated cell death

LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes) is a member of the tumor necrosis factor superfamily that can interact with lymphotoxin-beta receptor (LTbetaR), herpes virus entry mediator, and decoy receptor (DcR3). In our previous study, we showed that LIGHT is able to induce cell death via the non-death domain containing receptor LTbetaR to activate both caspase-dependent and caspase-independent pathway. In this study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar binding specificity as wild type LIGHT to LTbetaR, but lose the ability to interact with herpes virus entry mediator. By using both LIGHT-R228E and agonistic anti-LTbetaR monoclonal antibody, we found that signaling triggered by LTbetaR alone is sufficient to activate both caspase-dependent and caspase-independent pathways. Cross-linking of LTbetaR is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of caspase-3 activation, and kinase-inactive ASK1-KE mutant can inhibit LTbetaR-mediated cell death. This suggests that ASK1 is one of the factors involved in the caspase-independent pathway of LTbetaR-induced cell death.     

Stimulating lymphotoxin beta receptor on the dendritic cells is critical for their homeostasis and expansion

The increased number of dendritic cells (DCs) inside lymphoid tissue may contribute to the enhanced priming of lymphocytes. The homeostasis of splenic DCs has mostly been attributed to their migration to the spleen via the chemokine microenvironment induced by lymphotoxin beta receptor (LTbetaR) signaling on splenic stromal cells. In this study we show that the lack of direct LTbetaR signaling on DCs is associated with the reduction of the number of DCs in the spleen independently of chemokine gradients. LTbetaR-/- mice have reduced DCs and reduced BrdU incorporation on DCs, and fewer DCs from LTbetaR-/- mice are detected in the spleen. Furthermore, increased expression of LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpes virus entry mediator on T cells) on T cells, a member of the TNF family (TNFSF14) and a ligand for LTbetaR, could dramatically increase the number of T cells and DCs, which leads to severe autoimmune diseases in a LTbetaR-dependent fashion. In vitro, LIGHT could directly promote accumulation of bone marrow-derived DCs. Furthermore, intratumor expression of LIGHT can dramatically expand DCs in situ, and inoculation of DCs into tumor tissues enhanced tumor immunity. Therefore, LTbetaR signaling on DCs is required for their homeostasis during physiology and pathological conditions, and increased LIGHT-LTbetaR interaction could stimulate DC expansion for T cell-mediated immunity.     

Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells

Activation of the classical and noncanonical NF-kappaB pathways by ligation of the lymphotoxin (LT)-beta receptor (LTbetaR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTbetaR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTbetaR ligation on endothelial cells remain unclear. We therefore questioned whether LTbetaR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-kappaB-dependent gene expression. We demonstrate that the LTbetaR ligands LIGHT and LTalpha1beta2 activate both NF-kappaB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTalpha1beta2 and not TNF activated the noncanonical pathway. LIGHT and LTalpha1beta2 induced the expression of classical NF-kappaB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTbetaR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTalpha1beta2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IkappaB kinase alpha, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTbetaR ligation regulates gene expression in endothelial cells via both NF-kappaB pathways and we identify CXCL12 as a bona fide noncanonical NF-kappaB-regulated gene in these cells.     

Lymphotoxin-beta receptor activation by activated T cells induces cytokine release from mouse bone marrow-derived mast cells

Lymphotoxin-beta receptor (LTbetaR) signaling is known to play a key role in embryonic lymphoid organ formation as well as maintenance of lymphoid architecture. Activation of the LTbetaR is induced by either the heterotrimeric lymphotoxin-alpha(1)beta(2) (LTalpha(1)beta(2)) or the homotrimeric LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV gpD for herpes virus entry mediator, a receptor expressed by T lymphocyte). Both ligands are expressed on activated lymphocytes. As mast cells reside in close proximity to activated T cells in some inflammatory tissues, we examined the expression of LTbetaR on bone marrow-derived mast cells and asked whether the LTbetaR-ligand interaction would allow communication between mast cells and activated T cells. We found that mast cells express LTbetaR at the mRNA as well as at the protein level. To investigate LTbetaR-specific mast cell activation, the LTbetaR on BMMC from either wild-type or LTbetaR-deficient mice was stimulated with recombinant mouse LIGHT or agonistic mAbs in the presence of ionomycin. LTbetaR-specific release of the cytokines IL-4, IL-6, TNF, and the chemokines macrophage inflammatory protein 2 and RANTES was detected. Moreover, coculture of mast cells with T cells expressing the LTbetaR ligands also entailed the release of these cytokines. Interference with a specific LTbetaR inhibitor resulted in significant suppression of mast cell cytokine release. These data clearly show that LTbetaR expressed on mast cells can transduce a costimulatory signal in T cell-dependent mast cell activation.     

The lymphotoxin-beta receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells

LIGHT is a tumor necrosis factor (TNF) ligand superfamily member, which binds two known cellular receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HveA). LIGHT is a homotrimer that activates proapoptotic and integrin-inducing pathways. Receptor binding residues via LIGHT were identified by introducing point mutations in the A' --> A" and D --> E loops of LIGHT, which altered binding to LTbetaR and HveA. One mutant of LIGHT exhibits selective binding to HveA and is inactive triggering cell death in HT29.14s cells or induction of ICAM-1 in fibroblasts. Studies with HveA- or LTbetaR-specific antibodies further indicated that HveA does not contribute, either cooperatively or by direct signaling, to the death pathway activated by LIGHT. LTbetaR, not HveA, recruits TNF receptor-associated factor-3 (TRAF3), and LIGHT-induced death is blocked by a dominant negative TRAF3 mutant. Together, these results indicate that TRAF3 recruitment propagates death signals initiated by LIGHT-LTbetaR interaction and implicates a distinct biological role for LIGHT-HveA system.     

A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.

TR6 (decoy receptor 3 (DcR3)) is a new member of the tumor necrosis factor receptor (TNFR) family. TR6 mRNA is expressed in lung tissues and colon adenocarcinoma, SW480. In addition, the expression of TR6 mRNA was shown in the endothelial cell line and induced by phorbol 12-myristate 13-acetate/ionomycin in Jurkat T leukemia cells. The open reading frame of TR6 encodes 300 amino acids with a 29-residue signal sequence but no transmembrane region. Using histidine-tagged recombinant TR6, we screened soluble forms of TNF-ligand proteins with immunoprecipitation. Here, we demonstrate that TR6 specifically binds two cellular ligands, LIGHT (herpes virus entry mediator (HVEM)-L) and Fas ligand (FasL/CD95L). These bindings were confirmed with HEK 293 EBNA cells transfected with LIGHT cDNA by flow cytometry. TR6 inhibited LIGHT-induced cytotoxicity in HT29 cells. It has been shown that LIGHT triggers apoptosis of various tumor cells including HT29 cells that express both lymphotoxin beta receptor (LTbetaR) and HVEM/TR2 receptors. Our data suggest that TR6 inhibits the interactions of LIGHT with HVEM/TR2 and LTbetaR, thereby suppressing LIGHT- mediated HT29 cell death. Thus, TR6 may play a regulatory role for suppressing in FasL- and LIGHT-mediated cell death.     

The role of lymphotoxin receptor signaling in diseases

LT, LIGHT, and TNF are core family members of the TNFR superfamily of cytokines. LT and LIGHT, produced primarily by lymphocytes, interact with LTbetaR expressed by stromal and epithelial cells. Extensive studies over the last decade have revealed a critical role of LT-LTbetaR interactions for organogenesis and maintenance of the secondary lymphoid organs and in the generation of an efficient humoral immune response to various pathogens. LTbetaR's function beyond the lymphoid organs shows valuable potential yet remains largely undefined. Recent studies indicate that LTbetaR signaling is required for liver regeneration, hepatitis, and hepatic lipid metabolism. The balance of beneficial and detrimental effects of LTbetaR is critical for understanding the mechanisms of autoimmune disease and liver function and may open a new avenue for therapeutic intervention. This review will discuss recent advances in understanding LTbetaR's role in various human and murine disease models while focusing on its regulation of and implications in various liver related diseases.     

Activation of the lymphotoxin-beta receptor induces NFkappaB-dependent interleukin-6 and MIP-2 secretion in mouse fibrosarcoma cells

Activation of the lymphotoxin beta-receptor (LTbetaR), a member of the tumor necrosis factor receptor family, plays a crucial role in lymphoid organogenesis and tumor development. Lymphotoxin alpha(1)beta(2) (LTalpha(1)beta(2)) and LIGHT have been identified as membrane anchored ligands for the LTbetaR. While LTbetaR is expressed on a wide range of cell types e.g. fibroblasts and monocytes, the ligands are expressed only on activated lymphocytes and NK cells. In order to characterize LTbetaR expression and the biological consequences of LTbetaR activation rat anti-mouse LTbetaR monoclonal antibodies were generated. These antibodies recognized a mouse LTbetaR-Ig fusion protein as well as endogenous LTbetaR on a variety of mouse fibroblast and fibrosarcoma cell lines. Specificity was demonstrated by the lack of binding to LTbetaR-deficient embryonic fibroblasts. Competitive binding studies revealed that three different epitopes were recognized by the monoclonal antibodies. Two of the monoclonals activated the LTbetaR and induced activation of NFkappaB and secretion of MIP-2 and IL-6 in L929 mouse fibroblast cells. MIP-2 and IL-6 secretion was NFkappaB-dependent because IkappaB-transfected cells released significantly reduced amounts of both mediators.     

Stoichiometry of LTbetaR binding to LIGHT

LTbetaR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTalpha1beta2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTbetaRIgG, and monomeric LTbetaR protein binding to both LIGHT and LTalpha1beta2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTalpha1beta2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTbetaRIgG and LTbetaR binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC-LS corresponded to a single LTbetaRIgG bound to a LIGHT trimer, or two LTbetaR bound per LIGHT. Sedimentation velocity of varied ratios of LTbetaR to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LTbetaR or LTbetaRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTbetaR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFbeta, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor-ligand complexation paradigm for the entire TNF family. The LTbetaR/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LTbetaR rather than trimerization as expected from the structural paradigm.     

Targeting tumors with LIGHT to generate metastasis-clearing immunity

Metastatic diseases cause the majority of morbidity and mortality of cancer patients. Established tumors form both physical and immunological barriers to limit immune detection and destruction. Current immunotherapy of vaccination and adoptive transfer shows limited effect at least in part due to the existing barriers in the tumors and depending on the knowledge of tumor antigens. Tumor necrosis factor (TNF) superfamily (TNFSF) member 14 (TNFSF14) LIGHT interacts with stromal cells, dendritic cells (DCs), NK cells, na?ve and activated T cells and tumor cells inside the tumor tissues via its two functional receptors, HVEM and lymphotoxin beta receptor (LTbetaR). Targeting tumor tissues with LIGHT leads to augmentation of priming, recruitment, and retention of effector cells at tumor sites, directly or indirectly, to induce strong anti-tumor immunity to inhibit the growth of primary tumors as well as eradicate metastases. Intratumor treatment would break tumor barriers and allow strong immunity against various tumors without defining tumor antigens. This review summarizes recent findings to support that LIGHT is a promising candidate for an effective cancer immunotherapy.     

Signaling pathways of LIGHT induced macrophage migration and vascular smooth muscle cell proliferation

The biological actions of LIGHT, a member of the tumor necrosis factor superfamily, are mediated by the interaction with lymphotoxin-beta receptor (LTbetaR) and/or herpes virus entry mediator (HVEM). Previous study demonstrated high-level expressions of LIGHT and HVEM receptors in atherosclerotic plaques. To investigate the role of LIGHT in the functioning of macrophages and vascular smooth muscle cells (VSMC) in relation to atherogenesis, we determined the effects of LIGHT on macrophage migration and VSMC proliferation. We found LIGHT through HVEM activation can induce both events. LIGHT-induced macrophage migration was associated with activation of signaling kinases, including MAPKs, PI3K/Akt, NF-kappaB, Src members, and FAK. Proliferation of VSMC was also shown relating to the activation of MAPKs, PI3K/Akt, and NF-kappaB, which consequently led to alter the expression of cell cycle regulatory molecules. Down-regulation of p21, p27, and p53, and inversely up-regulation of cyclin D and RB hyper-phosphorylation were demonstrated. In conclusion, LIGHT acts as a novel mediator for macrophage migration and VSMC proliferation, suggesting its involvement in the atherogenesis.     

LIGHT Is critical for IL-12 production by dendritic cells, optimal CD4+ Th1 cell response, and resistance to Leishmania major

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT(-/-) mice were severely impaired in IL-12p40 production following IFN-gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LTbetaR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-gamma production in vivo. In an infection model, injection of HVEM-Ig or LTbetaR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTbetaR-Ig was also reproduced in LIGHT(-/-) --> RAG1(-/-) chimeric mice. In contrast, L. major-infected LTbeta(-/-) mice do not develop acute disease, suggesting that the effect of LTbetaR-Ig is not due to blockade of membrane LT (LTalpha1beta2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-gamma-producing CD4(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.     

Mechanism of LIGHT/interferon-gamma-induced cell death in HT-29 cells

LIGHT is a member of tumor necrosis factor (TNF) superfamily, and previous studies have indicated that in the presence of interferon-gamma (IFN-gamma), LIGHT through LTbetaR signaling can induce cell death with features unlike classic apoptosis. In present study, we investigated the mechanism of LIGHT/IFN-gamma-induced cell death in HT-29 cells, where the cell death was profoundly induced when sub-toxic concentrations of LIGHT and IFN-gamma were co-treated. LIGHT/IFN-gamma-induced cell death was accompanied by DNA fragmentation and slight LDH release. This effect was not affected by caspase, JNK nor cathepsin B inhibitors, but was partially prevented by p38 mitogen-activated protein kinase (MAPK) and poly (ADP-ribose) polymerase (PARP) inhibitors, and abolished by aurintricarboxylic acid (ATA), which is an inhibitor of endonuclease and STATs signaling of IFN-gamma. Immunobloting reveals that LIGHT/IFN-gamma could induce p38 MAPK activity, Bak and Fas expression, but down-regulate Mcl-1. Besides, LIGHT/IFN-gamma could not activate caspase-3 and -9, but decreased mitochondrial membrane potential. Although LIGHT could not affect IFN-gamma-induced STAT1 phosphorylation and transactivation activity, which was required for the sensitization of cell death, survival NF-kappaB signaling of LIGHT was inhibited by IFN-gamma. These data suggest that co-presence of LIGHT and IFN-gamma can induce an integrated interaction in signaling pathways, which lead to mitochondrial dysfunction and mix-type cell death, not involving caspase activation.     

The TNF receptor and Ig superfamily members form an integrated signaling circuit controlling dendritic cell homeostasis

Dendritic cells (DC) constitute the most potent antigen presenting cells of the immune system, playing a key role bridging innate and adaptive immune responses. Specialized DC subsets differ depending on their origin, tissue location and the influence of trophic factors, the latter remain to be fully understood. Myeloid-associated lymphotoxin-beta receptor (LTbetaR) signaling is required for the local proliferation of lymphoid tissue DC. This review focuses on the LTbetaR signaling cascade as a crucial positive trophic signal in the homeostasis of DC subsets. The noncanonical coreceptor pathway comprised of the immunoglobulin (Ig) superfamily member, B and T lymphocyte attenuator (BTLA) and TNFR superfamily member, herpesvirus entry mediator (HVEM) counter regulates the trophic signaling by LTbetaR. Together both pathways form an integrated signaling circuit achieving homeostasis of DC subsets.     

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.     

A novel vaccine containing EphA2 epitope and LIGHT plasmid induces robust cellular immunity against glioma U251 cells

EphA2 is a receptor tyrosine kinase and can be acted as an attractive antigen for glioma vaccines. In addition, LIGHT plays an important role on enhancing T cell proliferation and cytokine production. To improve the CTL mediated immune response against glioma cells, we prepared the novel vaccine containing EphA2_883–891 peptide (TLADFDPRV) and LIGHT plasmid and utilized it to immunize the HLA-A2 transgenic HHD mice. In addition, trimera mice were immunized with the novel vaccine to elicit the antitumor immune response. The results demonstrated that the novel vaccine could induce robust cellular immunity against glioma U251 cells without lysing autologous lymphocytes. Moreover, the novel vaccine could significantly inhibit the tumor growth and prolong the life span of tumor bearing mice. These findings suggested that the novel vaccine containing EphA2 epitope and LIGHT plasmid could induce anti-tumor immunity against U251 cells expressing EphA2, and provided a promising strategy for glioma immunotherapy.