Tumor Necrosis Factor Pretreatment Research Articles

Abstract # 1764 Tumor necrosis factor (TNF) increases transient potential receptor type ankyrin (TRPA)-1 expression and function in rheumatoid arthritis synovial fibroblasts: TRPA1 as therapeutic target to reduce joint inflammation in RA?

In collagen-induced arthritis, endocannabinoids, like anandamide, improves synovial inflammation. In previous studies anandamide decreased TNF-induced IL-6, IL-8 and MMP-3 in synovial fibroblasts (SFs) in a TRPA1-dependent manner. Joint pain and inflammation are driven by pro-inflammatory cytokines like TNF, which is also involved in sensitizing TRP-channels. Therefore, the hypothesis was tested that TNF augments TRPA1 mediated effects in SFs. TRPA1 expression after TNF exposure was analyzed by western blotting and cell based ELISA. Cell proliferation was determined by cell titer blue and cell death was monitored by lactate dehydrogenase (LDH) levels. Calcium flux assays were performed with Fura-2. Exposure of SFs to TNF (10 ng/ml) for 72 h increased TRPA1 protein accompanied by an increase in intracellular calcium following TRPA1 agonist allyl isothiocyanate (AITC). While SFs did not respond to AITC without TNF pretreatment, TNF pre-incubation elicited a robust increase in calcium in response to AITC (10 μ M–500 μ M). This was paralleled by increased LDH indicating cell death in response to TRPA1 activation. Cell proliferation was also modulated by TRPA1 activation. While TNF naive cells demonstrated reduced proliferation in response to 100 μ M AITC, TNF pre-treatment lowered the effective concentration to 12.5 μ M. The observed increase in TRPA1 mediated effects by TNF might explain lack of effects of endocannabinoids on basal cytokine production in SFs. Targeting TRPA1 in inflammation might have therapeutic advantages, since it mainly affects SFs pre-activated by pro-inflammatory cytokines.

Protective effect of tumor necrosis factor-alpha against subsequent endotoxemia in mice is mediated, in part, by interleukin-10.

Tumor necrosis factor (TNF)-alpha administration in large amounts can induce a state of shock similar to that observed in patients suffering from septic shock. Small doses of TNF-alpha induce only mild, transient hemodynamic alterations and can confer protection against subsequent inflammatory stimuli. The objective of this study was to determine whether this protective mechanism could be attributed to activity of the anti-inflammatory cytokine interleukin (IL)-10. Prospective, randomized, controlled study. Investigative intensive care unit at a medical university. Female BALB-c mice, 10-12 wks of age (approximately 20 g). All mice were subjected to intraperitoneal (ip) injection of lipopolysaccharide (LPS; Escherichia coli 0111:B4, 125 microg). Mice were randomly assigned to the following groups: TNF-alpha pretreated (100 microg ip 24 hrs before LPS); control (TNF vehicle alone 24 hrs before LPS); TNF/anti-IL-10 pretreated (TNF pretreatment as above and a neutralizing anti-IL-10 antibody); TNF/anti-IL-10 control (TNF pretreatment as above and an isotype-matched control antibody with no IL-10 activity); IL-10 (100 microg ip 1 hr before LPS); and IL-10 control (IL-10 vehicle 1 hr before LPS). Mice were observed for a 48-hr period after endotoxin administration. Mortality in each group was recorded. Separate groups of mice were pretreated with TNF (or vehicle) and killed at 0, 2, or 4 hrs after LPS injection for collection of serum and peritoneal lavage samples that were used to assay IL-10 concentrations. A small dose of TNF-alpha attenuated mortality in mice that were subsequently injected with a highly lethal dose of endotoxin and observed for 48 hrs. Peritoneal lavage fluid concentrations of IL-10 were consistently higher in TNF-pretreated mice after endotoxin administration. The TNF-alpha protective effect was reversed by administration of a neutralizing antibody directed against murine IL-10. These findings indicate that administration of a low dose of TNF-alpha can induce cross-tolerance to endotoxin by induction of endogenous anti-inflammatory mechanisms.

Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD

The cytokine tumor necrosis factor (TNF) is toxic to some mitotic cells, but protects cultured neurons from a variety of insults by mechanisms that are unclear. Pretreatment of neurons or astrocytes with TNF caused significant increases in MnSOD activity, and also significantly attenuated 3-nitropropionic acid (3-NP) induced superoxide accumulation and loss of mitochondrial transmembrane potential. In oligodendrocytes, however, MnSOD activity was not increased, and 3-NP toxicity was unaffected by TNF. Genetically engineered PC6 cells that overexpress MnSOD also were resistant to 3-NP-induced damage. TNF pretreatment and MnSOD overexpression prevented 3-NP induced apoptosis, and shifted the mode of death from necrosis to apoptosis in response to high levels of 3-NP. Mitochondria isolated from either MnSOD overexpressing PC6 cells or TNF-treated neurons maintained resistance to 3-NP-induced loss of transmembrane potential and calcium homeostasis, and showed attenuated release of caspase activators. Overall, these results indicate that MnSOD activity directly stabilizes mitochondrial transmembrane potential and calcium buffering ability, thereby increasing the threshold for lethal injury. Additional studies showed that levels of oxidative stress and striatal lesion size following 3-NP administration in vivo are increased in mice lacking TNF receptors.

TUMOR NECROSIS FACTOR PROMOTES MOTOR FUNCTIONAL RECOVERY IN CRUSHED PERIPHERAL NERVE

The potential benefits of tumor necrosis factor pretreatment in promoting motor functional recovery of peripheral nerve following low load crush injury were examined. Using a specially designed crush device, rat sciatic nerve was subjected to a low load crush injury of 2-h duration. Recombinant murine tumor necrosis factor and saline were intraperitoneally injected into the experimental and control animals, respectively, prior to nerve crushing. Subsequent motor function was evaluated at intervals by measurement of the sciatic functional index. There was significantly ( P < 0.05 to < 0.01) more rapid recovery in the tumor necrosis factor pretreated group as compared to the controls between day 14 and day 28. The sciatic functional index in the tumor necrosis factor group improved to −69.3 ± 5.3 at day 14 and to nearly normal at day 21. In contrast, the sciatic functional index in the control group was −95.5 ± 3.1% at day 14 and did not approach normal until day 42. Histological results paralleled the functional findings. The results suggest that tumor necrosis factor pretreatment has the potential to attenuate neurostructural damage and promote motor functional recovery in rat peripheral nerve. Copyright © 1996 Elsevier Science Ltd.

Tumor necrosis factor and lymphotoxin: protection against oxidative stress through induction of MnSOD.

Tumor necrosis factor (TNF) and lymphotoxin (LT) are related cytokines produced in response to infection or oxidative insults such as radiation. These cytokines bind to the same receptors and have pleiotropic effects on a variety of cell types. TNF or LT pretreatment, which can induce the synthesis of "protective" proteins such as mitochondrial manganese superoxide dismutase (MnSOD), protects animals from lethal doses of radiation or the chemotherapeutic drug doxorubicin. In contrast, TNF or LT pretreatment of tumor cells, which do not express MnSOD, results in sensitization to these insults. Therefore, radio- or chemoprotection of normal cells may act partially through enhanced expression of MnSOD. On the other hand, tumor sensitization may result from activation of "killing" proteins such as interleukin-1 beta converting enzyme (ICE) or other ICE-like proteases, possibly through TNF/LT-induced oxygen free radicals. In addition to their originally described anti-tumor activity, these cytokines may have new therapeutic indications in protecting normal cells while sensitizing tumor cells to radiation or chemotherapeutic drugs.

Tumor Necrosis Factor Induces Lipopolysaccharide Tolerance in a Human Adenocarcinoma Cell Line Mainly through the TNF p55 Receptor

This study demonstrates that lipopolysaccharide (LPS) mediates induction of transcription factor NF kappa B and activation of the cytomegalovirus (CMV) promoter-enhancer in the SW480 cell line. These cells do not express a functional membrane CD14. The LPS response in SW480 cells was weaker and markedly slower than the tumor necrosis factor (TNF) response. Pretreatment with TNF for 72 h inhibited both TNF, tumor necrosis factor receptor (TNFR) p55, TNFR p75, and LPS-mediated activation of nuclear factor -kappa B (NF kappa B), whereas pretreatment with LPS only inhibited the LPS response. TNFR p55 antibody pretreatment resulted in marked inhibition of the LPS response, while pretreatment with TNFR p75 antiserum only had a weak inhibitory effect. Flowcytometric analysis showed that LPS binding as well as expression of TNFR p55 and TNFR p75 were not affected by LPS or TNF pretreatment, indicating that the observed inhibition is not due to reduction of specific binding sites at the cell surface. The results suggest that LPS signaling in SW480 cells involves intracellular components which may be depleted or inactivated via TNFR p55, indicating that the LPS and TNFR p55 pathways overlap. We propose that TNFR p55 can mediate activation of NF kappa B and cytomegalovirus promoter-enhancer in SW480 cells via two distinct mechanisms, one which is activated only via TNFR p55 and leads to rapid activation of NF kappa B, and another which is overlapping with the LPS pathway.

Alterations of bacterial clearance induced by endotoxin and tumor necrosis factor

The purpose of the study was to investigate the potential influence of endotoxin and tumor necrosis factor (TNF) on immune function in terms of systemic clearance and organ distribution of injected Escherichia coli in a rabbit model. To enable quantification of the clearance process, defined numbers of exogenous E. coli (1.3 x 10(8) CFU) were injected intravenously 60 min after bolus application of TNF (4 x 10(5) U, n = 6), after infusion of endotoxin (40 micrograms/kg of body weight) for 1 h (n = 6) or 4 h (n = 6), or after saline infusion (controls, n = 6). Parameters monitored were arterial pressure, oxygen uptake, and rates of bacterial elimination from the blood. At 180 min after E. coli injection, the animals were sacrificed, and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts. Endotoxin infusion produced a significant delay in blood clearance compared with saline and TNF pretreatment. The diminished systemic bacterial elimination was associated with significantly higher numbers of E. coli in the organs, thus reflecting reticuloendothelial system dysfunction. TNF had no major influence on the elimination kinetics of bacteria but affected the tissue distribution pattern with increased accumulation of E. coli in the lung (up to 100-fold of control values; P < 0.001).

Role of tumor necrosis factor in oxygen toxicity.

mRNA from lungs of mice exposed to high-dose oxygen (greater than 95%) for 3 days demonstrated increased expression of the genes for tumor necrosis factor (TNF), interleukin-1, and interleukin-6 compared with mRNA from lungs of mice exposed to room air. Daily treatment of mice exposed to high-dose oxygen with an antibody to TNF improved survival compared with mice receiving a similar dose of control immunoglobulin G. Pretreatment of mice with repetitive sublethal intraperitoneal doses of recombinant human TNF for 3 days or a single intravenous dose followed by exposure to high-dose oxygen afforded a significant survival advantage compared with high-dose oxygen-exposed mice pretreated with vehicle or interleukin-1. The repetitive intraperitoneal TNF pretreatment reduced the development of interstitial pneumonitis, pulmonary edema, and lung weight gain associated with oxygen toxicity and enhanced expression of the gene for the free radical protective enzyme manganous superoxide dismutase in lung tissue, a gene that is augmented as mice are exposed to high-dose oxygen. Furthermore a single intravenous dose of TNF 24 h after oxygen exposure was still protective. The results suggest that the toxicity of oxygen therapy can be partially ameliorated by either treatment with anti-TNF antibody or pretreatment and early treatment with TNF. These findings are consistent with the hypothesis that oxygen exposure induces TNF, which causes part of the toxicity of high-dose oxygen, and that pretreatment or early treatment with TNF induces the gene for an enzyme that recently has been shown to be very effective in protecting mice from the toxicity of oxygen.

The in vitro antiviral activity of tumor necrosis factor (TNF) in WISH cells is mediated by IFN-β induction

We present evidence showing that TNF is capable of inducing an antiviral state in WISH cells thereby protecting them from the cytopathic effect of vesicular stomatitis virus. Establishment of the antiviral state requires pretreatment with TNF. Such pretreatment not only protects the cells in a dose-dependent manner, but it markedly reduces virus yield as well. Kinetic studies have shown that a pretreatment period as short as 4 h at 37°C is effective in conferring protection. The antiviral activity of TNF could be attributed to the induction of IFN-β. In fact, polyclonal antibodies to IFN-β completely neutralized the antiviral state elicited by TNF. 2–5A synthetase activity was significantly enhanced when the cells were treated with doses of TNF that afforded antiviral protection. Finally, addition of specific antibodies to IFN- β 2 (IL-6) during TNF pretreatment failed to abolish the antiviral state, thus suggesting that IFN- β 2 is not involved in the TNF-induced antiviral state. Also, a homogeneous IFN- β 2 preparation failed to exert antiviral activity in our cell system.

Two monokines, interleukin 1 and tumor necrosis factor, render cultured vascular endothelial cells susceptible to lysis by antibodies circulating during Kawasaki syndrome.

Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, IL-1, or tumor necrosis factor (TNF). Sera from 20 patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In-labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement-mediated killing of IL-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without IL-1 or TNF pretreatment. Expression of the IL-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that IL-1- and TNF-inducible endothelial target antigens are distinct from IFN-gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation.