Autocrine Tumor Necrosis Factor Research Articles

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Tackling poverty, treating obesity: a ‘whole system’ approach

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infants under 2 years old. Necroptosis has been implicated in the outcomes of respiratory virus infections. We report...

TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation.

The NOD-like receptor (NLR)-P3 inflammasome is a global sensor of infection and stress. Elevated NLRP3 activation levels are associated with human diseases, but the mechanisms controlling NLRP3 inflammasome activation are largely unknown. Here, we show that TGF-β activated kinase-1 (TAK1) is a central regulator of NLRP3 inflammasome activation and spontaneous cell death. Absence of TAK1 in macrophages induced spontaneous activation of the NLRP3 inflammasome without requiring toll-like receptor (TLR) priming and subsequent activating signals, suggesting a distinctive role for TAK1 in maintaining NLRP3 inflammasome homeostasis. Autocrine tumor necrosis factor (TNF) signaling in the absence of TAK1 induced spontaneous RIPK1-dependent NLRP3 inflammasome activation and cell death. We further showed that TAK1 suppressed homeostatic NF-κB and extracellular signal-related kinase (ERK) activation to limit spontaneous TNF production. Moreover, the spontaneous inflammation resulting from TAK1-deficient macrophages drives myeloid proliferation in mice, and was rescued by RIPK1 deficiency. Overall, these studies identify a critical role for TAK1 in maintaining NLRP3 inflammasome quiescence and preserving cellular homeostasis and survival.

MLKL-PITPα signaling-mediated necroptosis contributes to cisplatin-triggered cell death in lung cancer A549 cells

Necroptosis has been reported to be involved in cisplatin-induced cell death, but the mechanisms underlying the occurrence of necroptosis are not fully elucidated. In this study, we show that apart from apoptosis, cisplatin induces necroptosis in A549 cells. The alleviation of cell death by two necroptosis inhibitors—necrostatin-1 (Nec-1) and necrosulfonamide (NSA), and the phosphorylation of mixed lineage kinase domain-like protein (MLKL) at serine 358, suggest the involvement of receptor-interacting protein kinase 1 (RIPK1)-RIPK3-MLKL signaling in cisplatin-treated A549 cells. Additionally, the initiation of cisplatin-induced necroptosis relies on autocrine tumor necrosis factor alpha (TNF-α). Furthermore, we present the first evidence that phosphatidylinositol transfer protein alpha (PITPα) is involved in MLKL-mediated necroptosis by interacting with the N terminal MLKL on its sixth helix and the preceding loop, which facilitates MLKL oligomerization and plasma membrane translocation in necroptosis. Silencing of PITPα expression interferes with MLKL function and reduces cell death. Our data elucidate that cisplatin-treated lung cancer cells undergo a new type of programmed cell death called necroptosis and shed new light on how MLKL translocates to the plasma membrane.

Hyperosmotic stress enhances cytotoxicity of SMAC mimetics

Inhibitors of apoptosis (IAP) proteins contribute to cell death resistance in malignancies and emerged as promising targets in cancer therapy. Currently, small molecules mimicking the IAP-antagonizing activity of endogenous second mitochondria-derived activator of caspases (SMAC) are evaluated in phase 1/2 clinical trials. In cancer cells, SMAC mimetic (SM)-mediated IAP depletion induces tumor necrosis factor (TNF) secretion and simultaneously sensitizes for TNF-induced cell death. However, tumor cells lacking SM-induced autocrine TNF release survive and thus limit therapeutic efficacy. Here, we show that hyperosmotic stress boosts SM cytotoxicity in human and murine cells through hypertonicity-induced upregulation of TNF with subsequent induction of apoptosis and/or necroptosis. Hypertonicity allowed robust TNF-dependent killing in SM-treated human acute lymphoblastic leukemia cells, which under isotonic conditions resisted SM treatment due to poor SM-induced TNF secretion. Mechanistically, hypertonicity-triggered TNF release bypassed the dependency on SM-induced TNF production to execute SM cytotoxicity, effectively reducing the role of SM to TNF-sensitizing, but not necessarily TNF-inducing agents. Perspectively, these findings could extend the clinical application of SM.

T-3256336, a novel and orally available small molecule IAP antagonist, induced tumor cell death via induction of systemic TNF alpha production

Inhibitors of apoptosis proteins (IAPs) are a family of antiapoptotic regulators that have attracted attention as potential targets for cancer therapeutics. Although recent studies have revealed that small-molecule IAP antagonists induce tumor selective cell death in an autocrine tumor necrosis factor (TNF)α-dependent manner, the single-agent efficacy of IAP antagonists is restricted to a small subset of cancer cells. In this study, we showed that the single-agent activity of T-3256336 was limited to a few cancer cell lines in vitro, and these cell lines were defined by relatively high levels of TNFα mRNA expression. However, some other cancer cells, including PANC-1 cells, become drastically sensitive to T-3256336 when costimulated with exogenous TNFα. In PANC-1 mouse xenograft models, the administration of T-3256336 increased levels of several cytokines including TNFα and lead to tumor regression as a single agent, which was attenuated by the neutralization of circulating mouse TNFα with an antibody. These results suggest dual roles of IAP antagonists, increase systemic cytokines including TNFα, and sensitization of tumors to IAP antagonist-induced death.

Loss of the oncogenic phosphatase PRL-3 promotes a TNF-R1 feedback loop that mediates triple-negative breast cancer growth.

Stimulating tumor cell senescence and apoptosis are proven methods for therapeutically combating cancer. However, senescence and apoptosis are conventionally viewed as parallel, not sequential, processes. We have discovered that the metastasis-promoting phosphatase, PRL-3, is transcriptionally regulated by the NF-ĸB pathway in triple-negative breast cancer (TNBC) cells, and that PRL-3 knockdown elicits an autocrine tumor necrosis factor receptor 1 (TNF-R1) feedback loop that results in TNBC cell senescence followed by apoptosis. Knockdown of PRL-3 leads to rapid G1 cell cycle arrest and induction of a strong TNFα cytokine response that promotes a period of cellular senescence through TNF-R1-mediated activation of NF-ĸB. Senescent PRL-3 knockdown cells subsequently underwent apoptosis as a result of increased TNF-R1 signaling through the TNFα-associated extrinsic death pathway, shunting signaling away from the NF-ĸB cascade. These data suggest that TNF-R1 signaling dynamically re-programs after PRL-3 knockdown, from sustaining cell senescence through NF-ĸB to promoting apoptosis through TNF-R1 internalization and caspase-8 activation. The molecular mechanisms that determine the survival–death balance of TNF-R1 signaling are poorly understood, despite the fact that TNF-R1 has been extensively studied. Our results describe PRL-3 knockdown as a novel survival–death balance modifier of the TNF-R1 pathway, and show that senescent TNBC tumor cells can be sensitized to undergo apoptosis in a sequential manner.

The double life of RIPK1

ABSTRACTWe have recently identified receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1) as an oncogenic driver in melanoma in addition to its well-established role in controlling cell survival and death. Our studies show that RIPK1 promotes melanoma cell proliferation through a positive feedback loop of NFKB1-BIRC2/BIRC3-RIPK1 powered by autocrine tumor necrosis factor.

Network dynamics determine the autocrine and paracrine signaling functions of TNF

A hallmark of the inflammatory response to pathogen exposure is the production of tumor necrosis factor (TNF) that coordinates innate and adaptive immune responses by functioning in an autocrine or paracrine manner. Numerous molecular mechanisms contributing to TNF production have been identified, but how they function together in macrophages remains unclear. Here, we pursued an iterative systems biology approach to develop a quantitative understanding of the regulatory modules that control TNF mRNA synthesis and processing, mRNA half-life and translation, and protein processing and secretion. By linking the resulting model of TNF production to models of the TLR-, the TNFR-, and the NFκB signaling modules, we were able to study TNF's functions during the inflammatory response to diverse TLR agonists. Contrary to expectation, we predicted and then experimentally confirmed that in response to lipopolysaccaride, TNF does not have an autocrine function in amplifying the NFκB response, although it plays a potent paracrine role in neighboring cells. However, in response to CpG DNA, autocrine TNF extends the duration of NFκB activity and shapes CpG-induced gene expression programs. Our systems biology approach revealed that network dynamics of MyD88 and TRIF signaling and of cytokine production and response govern the stimulus-specific autocrine and paracrine functions of TNF.

Abstract 2278: The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway

Abstract Birinapant (TL32711), a SMAC-mimetic currently in clinical trials, antagonizes multiple members of the inhibitor of apoptosis (IAP) protein family to promote apoptosis in cancer cells through the formation of a caspase-8:RIPK1 complex and induction of autocrine tumor necrosis factor (TNF). Evidently, birinapant-resistant cells do not produce TNF upon drug treatment. However, the mechanism by which birinapant-sensitive cells produce TNF is not clear. Owing to the fact that mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes, we explored the role of MAPKs in birinapant-induced TNF production. Western blot analyses on a panel of birinapant-sensitive (SK-OV-3, EVSA-T, TOV-21G: all with an IC50 < 200nM) and birinapant-resistant (IGROV-1, HCT116, WIDR & SK-MEL-28: all with an IC50 > 10μM) cell lines showed that birinapant induced rapid and robust phosphorylation of p38MAPK in sensitive cells, which corresponded to increased TNF production as assessed by ELISA. However, resistant cells were not affected at multiple time points. The p38MAPK inhibitor, LY2228820, reduced the levels of birinapant-induced TNF (2-fold with 5nM birinapant after 24h) in birinapant-sensitive cancer cells (SK-OV-3), whereas inhibitors of Erk1/2 (PD98059) and JNK (SP600125) had no effect on TNF production. Interestingly, RNAi-mediated inhibition of p38α (MAPK14) isoform significantly inhibited birinapant-induced TNF production (∼3-fold with 5nM birinapant after 24h) in SK-OV-3 cells while inhibition of p38β (MAPK11) isoform upregulated TNF production (∼3-fold), suggesting that there may be a balance between p38MAPK isoforms to regulate TNF production. Pharmacological and RNAi-mediated inhibition of RIPK1 and caspase-8 activities in SK-OV-3 cells resulted in reduced birinapant-induced p38MAPK phosphorylation and TNF production. Furthermore, commercially available pooled siRNAs targeting different isoforms of cFLIP, a catalytically-inactive homologue of caspase-8, upregulated p38MAPK phosphorylation (∼4-fold) and TNF production (∼8 to 26-fold) in birinapant-resistant cancer cell lines (T24, HT1376 & IGROV-1), and conferred differential sensitivity to the drug treatment (T24-IC50: 7nM; HT1376-IC50: >10nM; IGROV-1-IC50: >1μM). Experiments involving cFLIP long (cFLIP-L) and short (cFLIP-S) isoform-specific siRNAs showed that inhibition of cFLIP-L, but not cFLIP-S, in T24 & HT1376 cells induced p38MAPK phosphorylation and TNF production, and conferred sensitivity to birinapant. However, the combined inhibition of cFLIP-L and cFLIP-S in IGROV-1 cells was required to induce p38MAPK phosphorylation, TNF production and apoptosis. Collectively, these observations indicate that birinapant-induced autocrine TNF production is triggered by the activation of the caspase-8/RIPK1/p38MAPK axis, and that cFLIP isoforms may confer a context-dependent negative regulatory effect. Citation Format: Gurpreet Singh Kapoor, Christopher A. Benetatos, Yasuhiro Mitsuuchi, Eric M. Neiman, Guangyao Yu, Mark A. Mckinlay, Jennifer Burns, John Silke, Stephen M. Condon, Srinivas K. Chunduru. The SMAC-mimetic birinapant regulates autocrine TNF production by caspase-8:RIPK1 complex via p38MAPK pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2278. doi:10.1158/1538-7445.AM2014-2278

CFLIP Regulates Skin Homeostasis and Protects against TNF-Induced Keratinocyte Apoptosis

FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIPfl/fl-K14CreERtam mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation.

Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production

AbstractDiseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4−/− or to Myd88−/− macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1−/−) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS.

Regulation of microglia effector functions by tumor necrosis factor signaling.

The exact biological role of the cytokine tumor necrosis factor (TNF) in the central nervous system (CNS) is not well understood; but overproduction of TNF by activated microglia has been implicated in neuronal death, suggesting that TNF inhibition in the CNS may be a viable neuroprotective strategy. We investigated the role of TNF signaling in regulation of microglia effector functions using molecular, cellular, and functional analyses of postnatal and adult microglia populations in the CNS. No differences were found by flow cytometric analyses in the basal activation state between TNF-null and wild-type mice. Although TNF-null microglia displayed an atypical morphology with cytoplasmic vacuoles in response to stimulation with lipopolysaccharide (LPS), the phagocytic response of TNF-null microglia to Escherichia coli particles in vitro was normal and there were no signs of enhanced caspase 3 activation or apoptosis. Functionally, conditioned media from LPS-stimulated TNF-null microglia was found to have significantly reduced levels of IL-10, IL-6, IL-1β, IL-12, and CXCL1 relative to wild-type microglia and exerted no cytotoxic effects on neurally differentiated dopaminergic (DA) MN9D cells. In contrast, incubation of wild-type microglia with TNF inhibitors selectively depleted the levels of soluble TNF and its cytotoxicity on MN9D cells. To distinguish whether reduced cytotoxicity by LPS-activated TNF-null microglia could be attributed to deficient autocrine TNF signaling, we employed primary microglia deficient in one or both TNF receptors (TNFR1 and TNFR2) in co-culture with MN9D cells and found that neither receptor is required to elicit LPS-evoked TNF production and cytotoxicity on DA cells.

Attenuating Smac mimetic compound 3‐induced NF‐κB activation by luteolin leads to synergistic cytotoxicity in cancer cells

Smac mimetics are potential anticancer therapeutics selectively killing cancer cells through autocrine tumor necrosis factor (TNF)-mediated apoptosis pathway. Our recent study reveal that the Smac mimetic compound 3 (SMC3)-activated NF-kappaB protects cancer cells against apoptosis, thus blunting SMC3's anticancer activity. Based on our previous observations that the nutrient flavonoid luteolin potently blocks TNF-induced NF-kappaB activation in cancer cells, we investigated if the combination of SMC3 and luteolin would achieve a synergistic anticancer activity. The results show that luteolin had no effect on autocrine TNF but it effectively blocked SMC3-induced nuclear factor kappa B (NF-kappaB) activation and expression of anti-apoptotic NF-kappaB targets. When SMC3 and luteolin were combined in treating cancer cells derived from lung and liver tumors, the activation of TNF-dependent apoptosis was markedly sensitized and a synergistic cytotoxic effect was achieved. In addition, the SMC3 and luteolin co-treatment had marginal effect on immortalized normal human bronchial epithelial cells. The results suggest that combination of SMC3 and luteolin is an effective approach for improving the anticancer value of SMC3, which has implications in cancer prevention and therapy.

IKKβ-mediated nuclear factor-κB activation attenuates smac mimetic–induced apoptosis in cancer cells

Smac mimetics (SM) have been recently reported to kill cancer cells through the extrinsic apoptosis pathway mediated by autocrine tumor necrosis factor (TNF). SM also activates nuclear factor-kappaB (NF-kappaB). However, how SM induces NF-kappaB and the role of NF-kappaB in SM-induced cancer cell death has not been well elucidated. We found that effective blockage of NF-kappaB had no detectable effect on SM compound 3 (SMC3)-induced TNF secretion, suggesting that the induction of TNF by SMC3 is independent of NF-kappaB. Conversely, SMC3-induced NF-kappaB activation was found to be mediated by autocrine TNF because this effect of SMC3 was effectively inhibited when TNF was blocked with either a TNF neutralizing antibody or TNF small interfering RNA. In addition, although SMC3 dramatically reduced c-IAP1 level, it had marginal effect on c-IAP2 expression, TNF-induced RIP modification, NF-kappaB activation, and downstream antiapoptosis NF-kappaB target expression. Furthermore, blocking NF-kappaB by targeting IKKbeta or RelA substantially potentiated SMC3-induced cytotoxicity, suggesting that the NF-kappaB pathway inhibits SMC3-induced apoptosis in cancer cells. Our results show that through TNF autocrine, SM induces an IKKbeta-mediated NF-kappaB activation pathway that protects cancer cells against SM-induced apoptosis, and thus, NF-kappaB blockage could be an effective approach for improving the anticancer value of SM.

Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster immune privilege and disease progression through PD-L1

Macrophages (Mφ) are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process of Mφ to trigger transient activation of monocytes in peritumoral stroma. We showed that a fraction of monocytes/Mφ in peritumoral stroma, but not in cancer nests, expresses surface PD-L1 (also termed B7-H1) molecules in tumors from patients with hepatocellular carcinoma (HCC). Monocytes activated by tumors strongly express PD-L1 proteins with kinetics similar to their activation status, and significant correlations were found between the levels of PD-L1+ and HLA-DRhigh on tumor-infiltrating monocytes. Autocrine tumor necrosis factor α and interleukin 10 released from activated monocytes stimulated monocyte expression of PD-L1. The PD-L1+ monocytes effectively suppressed tumor-specific T cell immunity and contributed to the growth of human tumors in vivo; the effect could be reversed by blocking PD-L1 on those monocytes. Moreover, we found that PD-L1 expression on tumor-infiltrating monocytes increased with disease progression, and the intensity of the protein was associated with high mortality and reduced survival in the HCC patients. Thus, expression of PD-L1 on activated monocytes/Mφ may represent a novel mechanism that links the proinflammatory response to immune tolerance in the tumor milieu.

Progesterone Inhibits HIV‐1 Replication in Human Trophoblast Cells through Inhibition of Autocrine Tumor Necrosis Factor Secretion

Progesterone levels are higher in placental barriers during pregnancy, but the effect of progesterone on human immunodeficiency virus type 1 (HIV-1) infection in placental cells has not been addressed. We hypothesize that progesterone may affect HIV infection. Purified trophoblastic cells and trophoblastic cell lines were infected or transfected with HIV-1, and the effect of progesterone was analyzed. Viral replication was measured by viral p24 or viral load quantification. Nuclear factor kappa -B (NF- kappa B) or long terminal repeat (LTR)-dependent transcription was measured by luciferase assays. Expression of chemokine receptors was analyzed by flow cytometry. Tumor necrosis factor (TNF) messenger RNA was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Progesterone inhibits HIV-1 replication in placental cells at the concentration found in the placental interface, at a postentry step, and does not affect cell surface expression of chemokine receptors. Progesterone did not inhibit basal or induced LTR transcription or NF- kappa B activation. TNF synthesis in placental cells is induced by HIV-1 infection that, in an autocrine manner, activates viral replication, because neutralizing anti-TNF antibodies block it. Progesterone inhibits the induction of TNF synthesis by viral infection and virus or gp-120-induced TNF transcription. Our results demonstrate that progesterone inhibits HIV-1 replication in placental cells by reducing TNF levels, which are required for optimal viral replication.

Autocrine Tumor Necrosis Factor Alpha Links Endoplasmic Reticulum Stress to the Membrane Death Receptor Pathway through IRE1α-Mediated NF-κB Activation and Down-Regulation of TRAF2 Expression

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

HIV-1 Infection Induces Differentiation of Immature Neural Cells through Autocrine Tumor Necrosis Factor and Nitric Oxide Production

Immature neural cell lines could be productively infected by HIV-1. Interestingly, this infection was associated with a differentiation to a mature neuronal phenotype, characterized by the expression of mature neurofilaments and cell adhesion molecules, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Infection also induced TNF-α and IL-1β mRNA expression, as well as the synthesis of inducible nitric oxide synthase by neuroblastoma cells. Exogenous addition of TNF-α, but not of IL-1β or many other cytokines, including nerve growth factor, mimicked those effects induced by infection. Moreover, blocking endogenous TNF-α or NO production in cultures of infected cells with a neutralizing anti-TNF-α antibody or inducible nitric oxide synthase inhibitors prevented the expression of the mature cell phenotype as well as expression of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. Addition of NO generators and TNF-α activated NF-κB- and intercellular cell adhesion molecule-1-dependent promoter transcription, whereas inducible nitric oxide synthase inhibitors prevented the transcriptional activation of intercellular cell adhesion molecule-1 promoter that was induced by TNF-α. Those results suggest that HIV can infect immature neural cells and this infection induces their neural development via a TNF-α- and NO-mediated mechanism.

Autocrine tumor necrosis factor (TNF) and lymphotoxin (LT) alpha differentially modulate cellular sensitivity to TNF/LT-alpha cytotoxicity in L929 cells.

Tumor necrosis factor (TNF) and lymphotoxin (LT) alpha are structurally and functionally related cytokines. We expressed the TNF and LT-alpha genes in murine fibrosarcoma L929r2 cells, which can be sensitized to TNF/LT-alpha-dependent necrosis by inhibitors of transcription or translation. Autocrine production of murine TNF in L929r2 cells completely downmodulated the expression of the 55- and 75-kD TNF receptors, resulting in resistance to TNF/LT-alpha cytotoxicity. Partial downmodulation of the 55-kD receptor was observed in human TNF-producing L929r2 cells. In contrast, an unaltered TNF receptor expression was found on LT-alpha L929r2 transfectants. Hence, although similar cytotoxic effects are induced by extracellularly administered TNF and LT-alpha, endogenous expression of these cytokines fundamentally differs in the way they modulate TNF receptor expression. Unlike LT-alpha, secreted by the classical pathway, TNF is first formed as a membrane-bound protein, which is responsible for receptor downmodulation. To explore whether the different pathways for secretion of TNF and LT-alpha explain this difference, we examined the effect of membrane-bound LT-alpha expression. This was obtained by exchange of the classical signal sequence of LT-alpha for the membrane anchor of chicken hepatic lectin. Membrane retention of LT-alpha resulted indeed in receptor downmodulation and TNF/LT-alpha resistance. We conclude that membrane retention of newly synthesized TNF or LT-alpha is absolutely required for receptor downmodulation and TNF/LT-alpha resistance.