Search

Soluble tumor necrosis factor-α (sTNF-α) plays an important role in colitis-associated cancer (CAC); however, little is known about transmembrane TNF-α (tmTNF-α). Here, we observed an increase in sTNF-α mainly in colitis tissues from an azoxymethane/dextran sodium sulfate (DSS)-induced CAC mouse model whereas tmTNF-α levels were chiefly increased on epithelial cells at the tumor stage. The ratio of intracolonic tmTNF-α/sTNF-α was negatively correlated with the levels of pro-inflammatory mediators (IL-1β, IL-6, and NO) and M1 macrophages but positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and the level of the anti-inflammatory cytokine IL-10, suggesting an anti-inflammatory effect of tmTNF-α. This effect of tmTNF-α was confirmed again by the induction of resistance to LPS in colonic epithelial cell lines NCM460 and HCoEpiC through the addition of exogenous tmTNF-α or transfection of the tmTNF-α leading sequence that lacks the extracellular segment but retains the intracellular domain of tmTNF-α. A tmTNF-α antibody was used to block tmTNF-α shedding after the first or second round of inflammation induction by DSS drinking to shift the time window of tmTNF-α expression ahead to the inflammation stage. Antibody treatment significantly alleviated inflammation and suppressed subsequent adenoma formation, accompanied by increased apoptosis. An antitumor effect was also observed when the antibody was administered at the malignant phase of CAC. Our results reveal tmTNF-α as a novel molecular marker for malignant transformation in CAC and provide a new insight into blocking the pathological process by targeting tmTNF-α processing.

Tumor necrosis factor-alpha (TNF-α) plays an important pathogenic role in cardiac hypertrophy and heart failure (HF); however, anti-TNF is paradoxically negative in clinical trials and even worsens HF, indicating a possible protective role of TNF-α in HF. TNF-α exists in transmembrane (tmTNF-α) and soluble (sTNF-α) forms. Herein, we found that TNF receptor 1 (TNFR1) knockout (KO) or knockdown (KD) by short hairpin RNA or small interfering RNA (siRNA) significantly alleviated cardiac hypertrophy, heart dysfunction, fibrosis, and inflammation with increased tmTNF-α expression, whereas TNFR2 KO or KD exacerbated the pathological phenomena with increased sTNF-α secretion in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy in vivo and in vitro, respectively, indicating the beneficial effects of TNFR2 associated with tmTNF-α. Suppressing TNF-α converting enzyme by TNF-α Protease Inhibitor-1 (TAPI-1) to increase endogenous tmTNF-α expression significantly alleviated TAC-induced cardiac hypertrophy. Importantly, direct addition of exogenous tmTNF-α into cardiomyocytes in vitro significantly reduced ISO-induced cardiac hypertrophy and transcription of the pro-inflammatory cytokines and induced proliferation. The beneficial effects of tmTNF-α were completely blocked by TNFR2 KD in H9C2 cells and TNFR2 KO in primary myocardial cells. Furthermore, we demonstrated that tmTNF-α displayed antihypertrophic and anti-inflammatory effects by activating the AKT pathway and inhibiting the nuclear factor (NF)-κB pathway via TNFR2. Our data suggest that tmTNF-α exerts cardioprotective effects via TNFR2. Specific targeting of tmTNF-α processing, rather than anti-TNF therapy, may be more useful for the treatment of hypertrophy and HF., In contrast to detrimental effects of soluble tumor necrosis factor-alpha (TNF-α) via TNFR1, this study shows that transmembrane TNF-α protects the heart by suppressing pressure overload-induced cardiac hypertrophy and inflammation via TNFR2. Targeting tmTNF-α processing may be more useful than TNF-antagonist for treatment of hypertrophy and heart failure.

// Meng Zhang 1, * , Jing Wang 1, * , Lingwei Jia 2 , Jin Huang 1 , Cheng He 1 , Fuqing Hu 2 , Lifei Yuan 1 , Guihua Wang 2 , Mingxia Yu 1 and Zhuoya Li 1 1 Department of Immunology, Basic Medical College, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, P.R. China 2 Molecular Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030, P.R. China * These authors have contributed equally to this work Correspondence to: Zhuoya Li, email: zhuoyali@mails.tjmu.edu.cn Keywords: activation-induced cell death, transmembrane TNF-α, secretory TNF-α, reverse signaling, apoptosis Received: June 29, 2016 Accepted: June 20, 2017 Published: July 10, 2017 ABSTRACT Secretory tumor necrosis factor-alpha (sTNF-α) is known to mediate activation- induced cell death (AICD). However, the role of tmTNF-α in AICD is still obscure. Here, we demonstrated that tmTNF-α expression significantly increased accompanied with enhanced apoptosis during AICD in Jurkat and primary human T cells. Knockdown or enhancement of tmTNF-α expression in activated T cells suppressed or promoted AICD, respectively. Treatment of activated T cells with exogenous tmTNF-α significantly augmented AICD, indicating that tmTNF-α as an effector molecule mediates AICD. As tmTNF-α can function as a receptor, an anti-TNF-α polyclonal antibody was used to trigger reverse signaling of tmTNF-α. This antibody treatment upregulated the expression of Fas ligand, TNF-related apoptosis-inducing ligand and tmTNF-α to amplify AICD, and promoted activated T cells expressing death receptor 4, TNF receptor (TNFR) 1 and TNFR2 to enhance their sensitivity to AICD. Knockdown of TNFR1 or TNFR2 expression totally blocked tmTNF-α reverse signaling increased sensitivity to sTNF-α- or tmTNF-α-mediated AICD, respectively. Our results indicate that tmTNF-α functions as a death ligand in mediation of AICD and as a receptor in sensitization of activated T cells to AICD. Targeting tmTNF-α in activated T cells may be helpful in facilitating AICD for treatment of autoimmune diseases.

Tumor necrosis factor alpha (TNF-α), originally described as an anti-neoplastic cytokine, has been found, in apparent contradiction to its name, to play an important role in promoting the development and progression of malignant disease. Targeting TNF-α with TNF antagonists has elicited an objective response in certain solid tumors in phase I and II clinical trials. This review focuses on the relationship of TNF-α expressed by leukemia cells and adverse clinical features of leukemia. TNF-α is involved in all steps of leukemogenesis, including cellular transformation, proliferation, angiogenesis, and extramedullary infiltration. TNF-α is also an important factor in the tumor microenvironment and assists leukemia cells in immune evasion, survival, and resistance to chemotherapy. TNF-α may be a potent target for leukemia therapy.

Transmembrane TNF-α (tmTNF-α) and secretory TNF-α (sTNF-α) display opposite effects in septic shock. Reducing tmTNF-α shedding can offset the detrimental effects of sTNF-α and increase the beneficial effect of tmTNF-α. We previously developed a monoclonal antibody that is specific for tmTNF-α and does not cross-react with sTNF-α. In this study, we show that this antibody can specifically suppress tmTNF-α shedding by competing with a TNF-α converting enzyme that cleaves the tmTNF-α ectodomain to release sTNF-α. This tmTNF-α antibody significantly inhibited LPS-induced secretion of interleukin (IL)-1β, IL-6, interferon-β, and nitric oxide by monocytes/macrophages, and protected mice from septic shock induced by lipopolysaccharide (LPS) or cecal ligation and puncture, while reducing the bacterial load. The mechanism associated with the protective effect of this tmTNF-α antibody involved promotion of LPS-induced toll-like receptor 4 (TLR4) internalization and degradation by recruiting Triad3A to TLR4. Moreover, the tmTNF-α antibody inhibited LPS-induced activation of nuclear factor-κB and interferon regulatory factor 3 pathways by upregulating expression of A20 and monocyte chemotactic protein-induced protein 1. Similarly, treatment of macrophages with exogenous tmTNF-α suppressed LPS/TLR4 signaling and release of proinflammatory cytokines, indicating that increased levels of tmTNF-α promoted by the antibody contributed to its inhibitory effect. Thus, use of this tmTNF-α antibody for specific suppression of tmTNF-α shedding may be a promising strategy to treat septic shock.

Tumor necrosis factor (TNF)-α exists in two bioactive forms, a 26-kDa transmembrane form (tmTNF-α) and a 17-kDa soluble form (sTNF-α). sTNF-α has been recognized as a key regulator of hepatitis; however, serum sTNF-α disappears in mice during the development of severe liver injury, and high levels of serum sTNF-α do not necessarily result in liver damage. Interestingly, in a mouse model of acute hepatitis, we have found that tmTNF-α expression on Kupffer cells (KCs) significantly increases when mice develop severe liver injury caused by lipopolysaccharide (LPS)/D-galactosamine (D-gal), and the level of tmTNF-α expression is positively related to the activity of serum transaminases. Therefore, we hypothesized that KC-expressed tmTNF-α constitutes a pathomechanism in hepatitis and have explored the role of tmTNF-α in this disease model. Here, we have compared the impact of KCs(tmTNFlow) and KCs(tmTNFhigh) on acute hepatitis in vivo and ex vivo and have further demonstrated that KCs(tmTNFhigh), rather than KCs(tmTNFlow), not only exhibit an imbalance in secretion of pro- and anti-inflammatory cytokines, favoring inflammatory response and exacerbating liver injury, but also induce hepatocellular apoptosis via tmTNF-α and the expression of another pro-apoptotic factor, Fas ligand. Our data suggest that KC(tmTNFhigh) is a major contributor to liver injury in LPS/D-gal-induced hepatitis.

Chemoresistance remains a major obstacle to successful treatment of breast cancer. Although soluble tumor necrosis factor-α (sTNF-α) has been implicated in mediating drug-resistance in human cancers, whether transmembrane tumor necrosis factor-α (tmTNF-α) plays a role in chemoresistance remains unclear. Here we found that over 50% of studied patients expressed tmTNF-α at high levels in breast cancer tissues and tmTNF-α expression positively correlated with resistance to anthracycline chemotherapy. Alteration of tmTNF-α expression changed the sensitivity of primary human breast cancer cells and breast cancer cell lines to doxorubicin (DOX). Overexpression of N-terminal fragment (NTF) of tmTNF-α, a mutant form with intact intracellular domain of tmTNF-α to transmit reverse signals, induced DOX-resistance. Mechanistically, the tmTNF-α/NTF-ERK-GST-π axis and tmTNF-α/NTF-NF-κB-mediated anti-apoptotic functions were required for tmTNF-α-induced DOX-resistance. In a xenograft mouse model, the combination of tmTNF-α suppression with chemotherapy significantly enhanced the efficacy of DOX. Our data indicate that tmTNF-α mediates DOX-resistance through reverse signaling and targeting tmTNF-α may be beneficial for the treatment of DOX-resistant breast cancer.

Tumor necrosis factor-alpha (TNF-α) exists in two forms: secretory TNF-α (sTNF-α) and transmembrane TNF-α (tmTNF-α). Although both forms of TNF-α induce tumor cell apoptosis, tmTNF-α is able to kill tumor cells that are resistant to sTNF-α-mediated cytotoxicity, indicating their differences in signal transduction. Here, we demonstrate that internalization of TNFR1 is crucial for sTNF-α- but not for tmTNF-α-induced apoptosis. sTNF-α induces binding of tumor necrosis factor receptor type 1-associated death domain protein (TRADD) to the death domain (DD) of TNFR1 and subsequent activation of nuclear factor kappa B (NF-κB), and the formation of death-inducing signaling complexes (DISCs) in the cytoplasm after internalization. In contrast, tmTNF-α induces DISC formation on the membrane in a DD-independent manner. It leads to the binding of signal transducer and activator of transcription 1 (STAT1) to a region spanning amino acids 319-337 of TNFR1 and induces phosphorylation of serine at 727 of STAT1. The phosphorylation of STAT1 promotes its binding to TRADD, and thus recruits Fas-associated protein with DD (FADD) and caspase 8 to form DISC complexes. This STAT1-dependent signaling results in apoptosis but not NF-κB activation. STAT1-deficiency in U3A cells counteracts tmTNF-α-induced DISC formation and apoptosis. Conversely, reconstitution of STAT1 expression restores tmTNF-α-induced apoptotic signaling in the cell line. Consistently, tmTNF-α suppresses the growth of STAT1-containing HT1080 tumors, but not of STAT1-deficient U3A tumors in vivo. Our data reveal an unappreciated molecular mechanism of tmTNF-α-induced apoptosis and may provide a new clue for cancer therapy.

It has been reported that TNFR2 is involved in regulatory T cell induction and myeloid-derived suppressor cell (MDSC) accumulation, two kinds of immunosuppressive cells contributing to tumor immune evasion. Because transmembrane TNF-α (tmTNF-α) is the primary ligand for TNFR2, we hypothesized that tmTNF-α is mainly responsible for the activation of MDSCs. Indeed, we found that tmTNF-α, rather than secretory TNF-α (sTNF-α), activated MDSCs with enhanced suppressive activities, including upregulating arginase-1 and inducible NO synthase transcription, promoting secretion of NO, reactive oxygen species, IL-10, and TGF-β, and enhancing inhibition of lymphocyte proliferation. This effect of tmTNF-α was mediated by TNFR2, as TNFR2 deficiency significantly impaired tmTNF-α–induced release of IL-10 and NO and inhibition of T cell proliferation by MDSC supernatant. Furthermore, tmTNF-α caused p38 phosphorylation and NF-κB activation, whereas inhibition of NF-κB or p38 with an inhibitor pyrrolidine dithiocarbamate or SB203580 abrogated tmTNF-α–mediated increased suppression of lymphocyte proliferation by MDSCs. Consistently, our in vivo study showed that ectopic expression of uncleavable tmTNF-α mutant by 4T1 cells significantly promoted tumor progression and angiogenesis, accompanied with more accumulation of MDSCs and regulatory T cells in the tumor site, increased production of NO, IL-10, and TGF-β, as well as poor lymphocyte infiltration. In contrast, enforced expression of sTNF-α mutant by 4T1 cells that only released sTNF-α without expression of surface tmTNF-α markedly reduced MDSC accumulation and induced more lymphocyte infiltration instead, showing obvious tumor regression. Our data suggest that tmTNF-α acts as a potent activator of MDSCs via TNFR2 and reveals another novel immunosuppressive effect of this membrane molecule that promotes tumor immune escape.

TNF antagonists may offer therapeutic potential in solid tumors, but patients who have high serum levels of TNF-α fail to respond to infliximab, suggesting consumption of the circulating antibody and loss of transmembrane TNF-α (tmTNF-α) on tumors by ectodomain shedding. Addressing this possibility, we developed a monoclonal antibody (mAb) that binds both full-length tmTNF-α and its N-terminal truncated fragment on the membrane after tmTNF-α processing but does not cross-react with soluble TNF-α. We documented high levels of tmTNF-α expression in primary breast cancers, lower levels in atypical hyperplasia or hyperplasia, but undetectable levels in normal breast tissue, consistent with the notion that tmTNF-α is a potential therapeutic target. Evaluations in vitro and in vivo further supported this assertion. tmTNF-α mAb triggered antibody-dependent cell-mediated cytotoxicity against tmTNF-α–expressing cells but not to tmTNF-α–negative cells. In tumor-bearing mice, tmTNF-α mAb delayed tumor growth, eliciting complete tumor regressions in some mice. Moreover, tmTNF-α mAb inhibited metastasis and expression of CD44v6, a prometastatic molecule. However, the antibody did not activate tmTNF-α–mediated reverse signaling, which facilitates tumor survival and resistance to apoptosis, but instead inhibited NF-κB activation and Bcl-2 expression by decreasing tmTNF-α–positive cells. Overall, our results established that tmTNF-α mAb exerts effective antitumor activities and offers a promising candidate to treat tmTNF-α–positive tumors, particularly in patients that are nonresponders to TNF antagonists. Cancer Res; 73(13); 4061–74. ©2013 AACR.

Immune tolerance at the fetomaternal interface must be established during the processes of implantation and pregnancy. Monoclonal nonspecific suppressor factor beta (MNSFβ) is a secreted protein that possesses antigen-nonspecific immune-suppressive function. It was previously reported that intrauterine immunoneutralization of MNSFβ significantly inhibited embryo implantation in mice. In the present study, MNSFβ protein expression was up- or downregulated by overexpression or RNA interference, respectively, in HCC-94 cells and the culture supernatants used to determine effects of MNSFβ on the secretion of IL-4 and TNFα from mouse lymphocytes as detected by ELISA. A coculture model of mouse embryos and endometrial stromal cells was also utilized to determine the effects of a specific anti-MNSFβ antibody on hatching and growth of embryos in vitro. The results show that MNSFβ induced secretion of IL-4 and inhibited secretion of TNFα from mouse lymphocytes. Following immunoneutralization of MNSFβ protein in the HCC-94 supernatant, the stimulatory effect of MNSFβ on IL-4 secretion from mouse lymphocytes was reduced, while the inhibitory effect on secretion of TNFα was abrogated. Expression of MNSFβ was detected in both embryonic and endometrial stromal cells, and its immunoneutralization inhibited the hatching and spreading of embryos in an in vitro coculture model. These results indicated that MNSFβ may play critical roles during the peri-implantation process by regulating cytokine secretion of lymphocytes and by mediating the crosstalk between embryonic cells and endometrial stromal cells.

Tumor necrosis factor alpha (TNF-α) has been implicated in the pathogenesis of Crohn's disease. TNF antagonists are effectively used to treat these patients, although the efficiency of different antagonists varies. In the present study we combined TNF-α binding cyclic peptide (TBCP) and TNFR1 binding cyclic peptide (TRBCP) to treat TNBS-induced colitis in rats for one week. The symptoms of colitis including bloody diarrhea, rectal prolapse, and a profound and sustained weight loss were significantly ameliorated and the colon inflammatory damage, both macroscopic and histological scores, MPO activity, and NO production were markedly decreased in rats by neutralization of TNF-α and blocking TNFR1, as compared with those in rats treated with irrelevant peptide or normal saline (P

Transmembrane tumor necrosis factor alpha (tmTNF-alpha) has a variety of biological activities different from soluble TNF-alpha (sTNF-alpha), but the only difference in sequence is its leader sequence (LS). To investigate the effect of the LS on tmTNF-alpha activity, single amino acid substitutions in the LS and its linked extracellular mature domain were made in an in vitro translation system and in an intact cell system. Mutations at Met(-71) and Cys(-28) in the LS obliterated cytotoxicity of tmTNF-alpha, whilst their secretory form retained full activity compared to parental sTNF-alpha. The lost cytotoxicity of Met(-71) mutant tmTNF-alpha was partly due to a reduced receptor binding activity. In spite of full receptor binding activity, Cys(-28) mutant tmTNF-alpha failed to induce NO production and iNOS mRNA transcription via forward signaling, but synergized with sTNF-alpha in IL-8 mRNA transcription via reverse signaling. The Asp(143) mutant tmTNF-alpha lost the ability to bind TNFR and to kill MCF-7 cells, whilst its secretory form retained about 60% cytotoxicity of parental sTNF-alpha. Although the mutation at Phe(87) had full activity in both forms, its membrane form induced a change in cell death mode from apoptosis to necrosis, in contrast to wild-type TNF-alpha whose membrane molecule chiefly induced apoptosis and secretory molecule mainly caused necrosis in MCF-7, respectively. The data suggest that the LS may be required for maintaining the correct structure and the bioactivity of tmTNF-alpha.

Interestingly, some lymphoma cells, expressing high levels of transmembrane (tm)TNF-alpha, are resistant to secretory (s)TNF-alpha-induced necrosis but sensitive to tmTNF-alpha-mediated apoptosis. As tmTNF-alpha mediates "forward" as well as "reverse" signaling, we hypothesize that a balanced signaling between forward and reverse directions may play a critical role in determining the fate of cells bearing tmTNF-alpha. Using Raji cells as a model, we first added exogenous tmTNF-alpha on fixed, transfected NIH3T3 cells onto Raji cells to examine tmTNF-alpha forward signaling and its effects, showing that constitutive NF-kappaB activity and cellular inhibitor-of-apoptosis protein 1 transcription were down-regulated, paralleled with Raji cell death. As Raji cells express tmTNF-alpha, an inhibition of their tmTNF-alpha expression by antisense oligonucleotide caused down-regulation of NF-kappaB activity. Conversely, increasing tmTNF-alpha expression by suppressing expression of TNF-alpha-converting enzyme that cleaves tmTNF-alpha led to an enhanced activation of NF-kappaB, indicating that tmTNF-alpha, but not sTNF-alpha, contributes to constitutive NF-kappaB activation. We next transfected Raji cells with a mutant tmTNF-alpha lacking the intracellular domain to competitively suppress reverse signaling via tmTNF-alpha; as expected, constitutive NF-kappaB activity was decreased. In contrast, treating Raji cells with sTNFR2 to stimulate reverse signaling via tmTNF-alpha enhanced NF-kappaB activation. We conclude that tmTNF-alpha, when highly expressed on tumor cells and acting as a receptor, promotes NF-kappaB activation through reverse signaling, which is helpful to maintain tumor cell survival. On the contrary, tmTNF-alpha, when acting as a ligand, inhibits NF-kappaB activity through forward signaling, which is inclined to induce tumor cell death.

TNF-alpha plays an important role in the pathogenesis of obesity and insulin resistance in which the effect of TNF-alpha signaling via TNF receptor type 1 (TNFR1) largely remains controversial. To delineate the role of TNFR1-mediated TNF-alpha signaling in the pathogenesis of this disorder, a TNFR1 blocking peptide-Fc fusion protein (TNFR1BP-Fc) was used for the present study. Wistar rats were fed a high-fat/high-sucrose (HFS) diet for 16 wk until obesity and insulin resistance developed. In comparison with increased body weight and fat weight, enlarged adipocytes, and hypertriglyceridemia in the obese state, the subsequent 4-wk treatment with TNFR1BP-Fc resulted in significant weight loss characterized by decreased fat pad weight and adipocyte size and reduced plasma triglycerides. Furthermore, obesity-induced insulin resistance, including hyperinsulinemia, elevated C-peptide, higher degree of hyperglycemia after glucose challenge, and less hypoglycemic response to insulin, was markedly improved, and the compensatory hyperplasia and hypertrophy of pancreatic islets were reduced. Interestingly, treatment with TNFR1BP-Fc markedly suppressed systemic TNF-alpha release and its local expression in pancreatic islets and muscle and adipose tissues. In addition, blockage of TNFR1-mediated TNF-alpha signaling in obese rats significantly enhanced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in the muscle and fat tissues. Our results strongly suggest a pivotal role for TNFR1-mediated TNF-alpha signaling in the pathogenesis of obesity and insulin resistance. Thus, TNFR1BP-Fc may be a good candidate for the treatment of this disease.

BACKGROUND Restoration of blood flow to the ischemic liver lobes may paradoxically exacerbate tissue injury, which is called hepatic ischemia/reperfusion injury (IRI). Toll-like receptor 4 (TLR4), expressed on several liver cell types, and the nuclear factor-kappa B (NF-kappaB) signaling pathway are crucial to mediating hepatic inflammatory response. Because IRI is essentially a kind of profound acute inflammatory reaction evoked by many kinds of danger signals, we investigated TLR4/NF-kappaB signaling pathway activation in a murine model of partial hepatic IRI. METHODS Wild-type mice (WT, C3H/HeN) or TLR4 mutant mice (C3H/HeJ) were subjected to 45 minutes of partial hepatic ischemia followed by 1 hour, 3 hours of reperfusion. Sham group accepted the same procedure without the obstruction of blood supply. At the end of reperfusion, the compromise of liver function and the histological change of liver sections were measured as the severity of liver injury. The level of endotoxin in the portal vein was measured by limulus assay. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). The levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in systemic blood after hepatic IRI were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS The compromise of liver function and the morphological injuries in mutant mice were relieved more markedly than those in WT mice after partial hepatic IRI. NF-kappaB activation in WT mice was stronger than that in TLR4 mutant mice, and both were stronger than those in the sham operated mice (P < 0.01). Endotoxin in each group was undetectable. The levels of TNF-alpha and IL-1beta in systemic blood were elevated in both strains, but lower in the sham operated group. These mediators were significantly decreased in TLR4 mutant mice compared with those in WT mice (P < 0.01). CONCLUSIONS The TLR4/NF-kappaB signaling pathway may mediate hepatic IRI triggered by endogenous danger signals. Inhibition of the TLR4/NF-kappaB pathway may be a potential therapeutic target for attenuating ischemia/reperfusion-induced tissue damage in some clinical settings.

To design an effective antibody therapy to improve clinical outcomes in leukemia, the identification of novel cell surface antigens is needed. Herein, we demonstrate a role for transmembrane tumor necrosis factor-α (tmTNF-α) in leukemia. To characterize tmTNF-α expression in acute leukemia (AL), normal hematopoietic cells, and nonhematopoietic tissues, we used a monoclonal antibody, termed C1, which specifically recognizes the tmTNF-α domain. We found that tmTNF-α was preferentially expressed by AL and leukemia stem cells (LSCs). More abundant expression correlated with poor risk stratification, extramedullary infiltration, and adverse clinical parameters. Moreover, knockdown of tmTNF-α(+) expression rendered leukemia cells more sensitive to chemotherapy in vitro and delayed regeneration of leukemia in NOD-SCID mice. Targeting tmTNF-α by C1 resulted in leukemia cell killing via antibody-dependent cell-mediated and complement-dependent cytotoxicity in vitro and inhibited leukemia cell growth in vivo while simultaneously sparing normal hematopoietic cells. Notably, C1 administration impaired the regeneration of leukemia in secondary serial transplantation into NOD-SCID mice. In conclusion, tmTNF-α has a favorable AL- and LSC-associated expression profile and is important for the survival and proliferation of these cells. C1-mediated targeting shows potent anti-LSC activity, indicating that tmTNF-α represents a novel target antigen in AL.

Objective To determine the region of human transmembrane tumor necrosis factor-alpha (TM-TNFα), essential for cytotoxic activity against human breast cancer cell line MCF-7.

To study the role of β-actin in the signaling of transmembrane TNF-α (tmTNF-α)-mediated cytotoxicity, we mainly used bioassay and apoptosis assay, detection of the visual changes in β-actin and intracellular translocation of signal molecules in response to tmTNF-α, and analysis of the signal molecules coupled or uncoupled with TNFR2 complex. These protocols might also be used to investigate the signaling events mediated by other transmembrane cytokines or members of TNF superfamily, which have transmembrane and soluble forms, as well as the involvement of actin cytoskeleton in signal transduction pathways.

Transmembrane TNF-alpha (tmTNF-alpha) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-alpha (tmTNF-alpha) at high level was resistant to soluble TNF-alpha (sTNF-alpha)-induced cytotoxicity, accompanied by constitutive NF-kappaB activation. In contrast, MCF-7 cells expressing tmTNF-alpha at very low level were sensitive to sTNF-alpha-induced cell death and had no detectable NF-kappaB activation. Consistently, siRNA-mediated tmTNF-alpha knockdown blocked NF-kappaB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-alpha, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-alpha containing LS constitutively activated NF-kappaB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-alpha lacking the cytoplasmic segment of LS neither activated NF-kappaB nor affected the sensitivity. However, NF-kappaB inhibitor PDTC suppressed NF-kappaB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-kappaB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)(2)-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-kappaB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-kappaB and resistance to sTNF-alpha-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-alpha-induced resistance in the breast cancer cells.

Transmembrane tumor necrosis factor-alpha (mTNF-alpha) is known to be the precursor of soluble TNF-alpha (sTNF-alpha). mTNF-alpha can act as a ligand on the TNF receptor- (TNFR)- bearing cell through 'forward signaling' or as a receptor on the TNF producing cell through 'reverse signaling'. In the current study, we investigated the role of mTNF-alpha-mediated reverse signaling in regulating sTNF-alpha-induced activation of human monocytic U937 cells. We demonstrated that pretreatment with sTNFRI, for inducing reverse signaling through mTNF-alpha, sensitized U937 cells to sTNF-alpha stimulation, as evidenced by an increase in reactive oxygen production and mRNA levels of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-8) in these cells. Further experiments revealed that IkappaB-alpha degradation was increased in the monocytic cells primed with sTNFRI, implying that reverse signaling of mTNF-alpha sensitizes U937 cells via an NF-kappaB-dependent mechanism. On the other hand, binding of sTNFRI to mTNF-alpha after sTNF-alpha-induced activation of U937 cells reduced mRNA stability (half-life) of IL-1beta and IL-8. The involvement of reverse signaling in the process was verified by using a mutated form of mTNF-alpha lacking the majority of the cytoplasmic domain. Our results clearly showed that enhanced mRNA degradation of the cytokines occurred only in U937 cells transfected with a wild-type mTNF-alpha, but not in those cells transfected with the mutant mTNF-alpha. Taken together, these data suggest that reverse signaling through mTNF-alpha may exert a double role in modulating sTNF-alpha bioactivity. It is positive when reverse signaling occurs prior to sTNF-alpha stimulation, while it is negative when reverse signaling occurs after the sTNF-alpha signal. Thus, our findings strengthen a role of mTNF-alpha-mediated reverse signaling in the regulation of immune-inflammatory response and control of inflammatory reaction.

The killing of tumour cells that are resistant to soluble TNF-alpha (sTNF-alpha) by the membrane-bound form of TNF-alpha (mTNF-alpha) suggests that different intracellular signalling pathways are involved. We found that mTNF-alpha induced apoptosis in HL-60 cells and failed to cause degradation of inhibitor of kappa B alpha (IkappaB-alpha) and translocation and activation of nuclear factor kappa B (NF-kappaB), whereas sTNF-alpha failed to induce apoptosis, but lowered cytoplasmic inhibitor of kappa B alpha, induced translocation of NF-kappaB to the nucleus and experimentally increased activity of the regulated luciferase. Furthermore, mTNF-alpha upregulated the expression of TNF receptor associated factor (TRAF) 1 and failed to induce TRAF1 and TRAF2 membrane translocation, but led to cytoplasmic colocalization. In contrast, sTNF-alpha stimulated the expression of TRAF1 and TRAF2, recruiting both molecules onto the cell membrane poststimulation. These results suggest that the increased susceptibility of HL-60 cells to mTNF-alpha may be due to the failure of TRAF2 membrane translocation caused by the upregulation of TRAF1 expression and formation of a TRAF1/TRAF2 complex in the cytoplasm, thereby inhibiting NF-kappaB activation and inducing apoptosis.

The proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) exists naturally in two forms, a 26 kDa transmembrane form (TM-TNFalpha), and a 17 kDa secretory form (S-TNFalpha). The biological roles for each of these forms of TNFalpha in tumor killing have not been completely elucidated. Therefore, in this study, three different recombinant retroviral vectors, wild-type TNFalpha, solely secretable TNFalpha mutant, and uncleavable transmembrane TNFalpha mutant, were constructed by molecular techniques and stably transfected into a murine hepatic carcinoma cell line (H22). TNFalpha, either secreted in cell culture supernatants by secretable TNFalpha mutant- or wild-type TNFalpha-producing tumor cells, or as a treansmembrane form expressed on the cell surface of uncleavable TNFalpha mutant- or wild-type TNFalpha-synthesizing tumor cells, was demonstrated to be cytotoxic against the TNF sensitive L929 cell line. The H22 cells transfected with the three different forms of TNFalpha were shown to kill parental H22 cells in an in vitro cytotoxicity assay [effect/target (E/T) ratio-dependent manner], and their maximal killing rates were approximately 38-43% at E/T ratio of 5:1. The injection of total 2.5 x 10(5) mixed cells containing transfected and parental H22 tumor cells at different ratios into syngeneic mice resulted in the inhibition of tumor growth with a maximal inhibition rates of approximately 57 approximately 72% at E/T ratio of 5:1. A transient weight loss was found in mice bearing solely secretable TNFalpha mutant producing tumors, whereas no obvious side effects were seen in mice bearing uncleavable TNFalpha mutant or wild-type TNFalpha expressing tumors. Finally, we demonstrate that tumors secreting S-TNFalpha promoted the subsequent infiltration of CD4(+) T cells, and to a lesser extent CD8(+) T cells, to the tumor site. The TM-TNFalpha expressing tumors up-regulated Fas (CD95) expression and inhibited the expression of tumor metastasis associated molecule CD44v3. These results suggest that S-TNFalpha and TM-TNFalpha kill cancer cells in vivo through different mechanisms of action. We conclude that the non-secreted form of TNFalpha may be an ideal candidate for cancer gene therapy due to its therapeutic potential and lowered side effect profile.

Tumor necrosis factor-alpha (TNF-a) exists naturally in two forms, transmembrane and secretory form. In the present study, three recombinant retroviral vectors containing human wild type TNF-a (Wt-TNF), secretory TNF-a (S-TNF, a mutant replaced its signal peptide by that of IL-2), and transmembrane TNF-a (TM-TNF, a mutant deleted a site for cleavage of TNF into the secretory form) were constructed respectively and transfected stably into murine hepatic carcinoma cell line (H22). TNF-a either secreted in supernatants by S-TNF and Wt-TNF producing tumor cells or expressed on cell surface of TM-TNF and Wt-TNF synthesizing tumor cells was demonstrated to be cytotoxic against TNF sensitive L929 cells. Also, the H22 cells transfected with three types of TNF-a can kill parental H22 in effect/target (E/T) dependent manner in vitro and their maximal killing rates were about 38|[sim]|43% at E/T ratio of 5:1. Injection of total 2.5|[prime]|105 cells mixed with transfected and parental H22 tumor cells at different ratios into syngeneic mice resulted in the inhibition of tumor growth and their maximal inhibition rates were about 57|[sim]|72% at E/T 5:1. A transient weight loss was found in mice bearing S-TNF producing tumors, whereas no obvious side effects were seen in mice bearing TM-TNF or Wt-TNF expressing tumors. It has been shown that S-TNF secreting tumor promoted significantly lymphocytes infiltration and attracted more CD4+ T cells than CD8+ T cells to the tumor site, while TM-TNF expressing tumor stimulated Fas expression and inhibited a tumor metastasis associated molecule CD44v3 expression on tumor cells. These results suggest that S-TNF and TM-TNF kill cancer cells in vivo through different mechanisms and that transmembrane form of TNF-a might be a safer candidate for tumor gene therapy due to its less side effects and retainable antitumor activity.