Transmembrane TNF Research Articles

Transmembrane TNF Is Sufficient to Initiate Cell Migration and Granuloma Formation and Provide Acute, but Not Long-Term, Control ofMycobacterium tuberculosisInfection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.

LPS resistance in monocytic cells caused by reverse signaling through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway.

The transmembrane form of tumor necrosis factor (mTNF), expressed on activated monocytes (MO) and macrophages (MPhi), is able to induce apoptosis in human endothelial cells (EC). Apoptosis is mediated by two distinct mechanisms: direct cell contact and a yet-unidentified soluble protein, death factor X. In addition, mTNF acts as a receptor that transduces a "reverse signal" into MO/MPhi when bound to the TNF receptor on EC. Reverse signaling by mTNF confers resistance to bacterial lipopolysaccharide (LPS). Stimulation of reverse signaling by mTNF blocks the ability of MO/MPhi to produce death factor X and proinflammatory cytokines. We have investigated which signaling pathways are used by mTNF acting as receptor. Reverse signaling triggers two independent pathways that can be distinguished by protein kinase C (PKC) inhibitors. The suppression of LPS-induced death factor X is dependent on PKC, whereas the suppression of LPS-mediated cytokine release is not. LPS and reverse signaling stimulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. It is interesting that the activation of reverse signaling by mTNF renders MO/MPhi refractory to a subsequent activation of the MAPK/ERK pathway by LPS. Thus, reverse signaling achieves LPS resistance in monocytic cells through interference with key signal-transduction pathways.

Exclusive Action of Transmembrane TNF in Adipose Tissue Leads to Reduced Adipose Mass and Local But Not Systemic Insulin Resistance

Exclusive Action of Transmembrane TNF in Adipose Tissue Leads to Reduced Adipose Mass and Local But Not Systemic Insulin Resistance

Exclusive action of transmembrane TNF alpha in adipose tissue leads to reduced adipose mass and local but not systemic insulin resistance.

Aberrant TNF alpha expression in adipocytes is a molecular mechanism by which insulin action is modulated in adipose tissue. While this might be a compensatory response to limit adipose expansion, neither the mechanisms underlying this local effect nor its systemic biological consequences have been studied. It is also not clear whether TNF alpha-induced insulin resistance in adipocyte alone is responsible for systemic insulin resistance in the absence of obesity. In a transgenic mouse model deficient in endogenous TNF alpha, we demonstrate that specific expression of the transmembrane TNF alpha (mTNF alpha) in adipocytes leads to decreased whole body adipose mass, and local, but not systemic insulin resistance. These data demonstrate that exclusive action of TNF alpha in adipose tissue strongly inhibits insulin action at this site and leads to reduced adiposity in mice. However, this isolated adipocyte insulin resistance in the context of reduced fat mass and/or the absence of obesity is insufficient to alter systemic glucose homeostasis.

Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells.

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.

Transmembrane TNF (pro-TNF) is palmitoylated

Human transmembrane tumor necrosis factor (pro-TNF) was examined for protein acylation. The cDNA encoding pro-TNF was expressed in both COS-1 cells and Sf9 cells and metabolic labeling with [ 3H]myristic or [ 3H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [ 3H]palmitic acid. The [ 3H]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site-directed mutagenesis of the two cysteine residues residing in the leader sequence of pro-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains of pro-TNF. Thus, pro-TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.

Tumour necrosis factor-alpha and transforming growth factor-beta are significantly associated with better prognosis in non-small cell lung carcinoma: putative relation with BCL-2-mediated neovascularization.

Recent in vivo and in vitro studies have demonstrated a wide spectrum of biologic activities of cytokines in the pathogenesis and progression of malignancy. Tumour necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) have emerged as two of the many host-derived mediators that seem to interfere with both antiproliferative and tumorigenic effects in malignant tumours including lung cancer. However, their association with tumour prognosis or prognostic factors has not yet been completely clarified. In this study, we assessed TNF-α and TGF-β mRNA expression by RT-PCR technique in 61 NSCLC samples, demonstrating the presence of TNF-α and TGF-β mRNA in 55.74% and 45.9% of cases, respectively. We also evaluated the expression of the two distinct transmembrane TNF receptors. TNFR-I and TNFR-II, with a PCR-positive signal in 70.49% and 65.57% of cases, respectively. In 49 of the 61 cases, we evaluated the prognostic impact of the two growth-inhibiting factors using the Kaplan–Meier analysis. In the univariate analysis patients without nodal metastatic involvement (P = 0.02), less advanced tumour stage (P = 0.02) or TNF-α and TGF-β positive cancers (P = 0.01 and P = 0.03) showed a favourable prognosis in terms of overall survival. Since our previous studies demonstrated a significant association between NSCLC behaviour, neoangiogenesis and bcl -2 expression, we investigated the putative relation between TNF-α and TGF-β on the one hand, and vascular count (as a measure of tumour angiogenesis) and bcl -2 protein expression, on the other hand. Our results showed a significant direct association between TNF-α and bcl -2 (P = 0.05) and an inverse association between TNF-α and microvessel count (P = 0.03). Moreover, as previously demonstrated, we observed a significant inverse correlation between bcl -2 protein expression and vascular count (P = 0.05), suggesting that the favourable effect of TNF-α on clinical outcome may be related to a bcl -2-mediated low neovascular development. © 2000 Cancer Research Campaign

Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages.

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-alpha (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MPhi), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MPhi with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MPhi as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.

Levels of Tumor Necrosis Factor-α (TNF-α) and Soluble TNF Receptors in Chronic Venous Leg Ulcers – Correlations to Healing Status

This study tested the hypothesis that excessive tumor necrosis factor-alpha (TNF-alpha) levels in chronic venous leg ulcers are associated with impaired healing. TNF-alpha was measured by two enzyme-linked immunosorbent assays and a bioassay (KYM-1D4) in paired wound fluid samples collected during the nonhealing and healing phases from 21 human patients with venous leg ulcers. Soluble TNF receptor levels (p55 and p75) were also measured. The levels of immunoreactive TNF-alpha were significantly higher in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers (p < 0.005), whereas the levels of bioactive TNF-alpha were not. Statistical analysis confirmed that TNF-alpha bioactivity relative to the amount of immunoreactive TNF-alpha was downregulated in wound fluid from nonhealing ulcers compared with healing ulcers. The levels of soluble p55 and p75 receptors in wound fluid showed a significant linear correlation (p < 0.001), suggesting a partially coordinated or common regulatory mechanism for the cleavage of transmembrane TNF receptors in chronic venous ulcers in vivo. Although the levels of soluble p75 receptors were significantly higher in nonhealing wound fluid compared with healing wound fluid (p < 0.025), these levels were theoretically inadequate to substantially neutralize the bioactivity of the accompanying TNF-alpha levels on their own. The bioactivity accompanying the elevated levels of immunoreactive TNF-alpha in wound fluid from nonhealing ulcers may have been further down-modulated by an additional mechanism. Because healing was initiated without a significant decline in the level of bioactive TNF-alpha, TNF-alpha-mediated events may not be the key events contributing to the impaired healing seen in chronic venous ulcers.

TNF- α transgenic and knockout models of CNS inflammation and degeneration

Tumour necrosis factor- α (TNF- α) plays a central role in inflammatory events including those taking place in the central nervous system (CNS), and has been implicated as a key pathogenicc mediator in several human inflammatory, infectious and autoimmune CNS disorders. Using transgenic and gene knockout mice we have investigated the role of deregulated TNF- α production in the CNS. We show that the overexpression of wild-type murine or human TNF- α transgenes by resident CNS astrocytes or neurons in sufficient to trigger a neurological disorder characterised by ataxia, seizures and paresis, with histopathological features of chronic CNS inflammation and white matter degeneration. Furthermore, we show that transmembrane human TNF- α is sufficient to trigger CNS inflammation and degeneration when overexpressed by astrocytes but not by neurons, indicating that target cells mediating the neuroinflammatory activities of TNF- α localise in the vicinity of astrocytes rather than neurons. Our results establish that both soluble and transmembrane molecular forms of TNF- α can play critical roles in vivo in the pathogenesis of CNS inflammation and demyelination, and validate TNF- α transgenic and mutant mice as important models for the further study of related human CNS diseases.

Astrocyte-specific but not neuron-specific transmembrane TNF triggers inflammation and degeneration in the central nervous system of transgenic mice.

To study the involvement of TNF in cerebral pathology in vivo and to define cellular interactions within the central nervous system (CNS) that promote TNF signaling, we have expressed this cytokine either as a wild-type or a mutant transmembrane form in astrocytes or neurons of transgenic mice. Mice expressing wild-type human TNF in either of these cell types spontaneously develop a neurologic disorder manifested by ataxia, seizures, and paralysis and bear histologic evidence of chronic CNS inflammation and degeneration. Moreover, astrocyte-specific expression of transmembrane TNF triggers a similar neurologic phenotype. Interestingly, transgenic mice producing a high level of transmembrane TNF in their neurons develop no apparent phenotypic abnormalities, suggesting that appropriate cellular interactions should form to allow for contact-dependent TNF signals to induce CNS pathology. These results demonstrate that target cells mediating the neuroinflammatory activities of TNF localize in the vicinity of astrocytes rather than neurons.