Tumor necrosis factor-alpha inhibits myogenesis through redox-dependent and -independent pathways

Muscle wasting accompanies diseases that are associated with chronic elevated levels of circulating inflammatory cytokines and oxidative stress. We previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) inhibits myogenic differentiation via the activation of nuclear factor-kappaB (NF-kappaB). The goal of the present study was to determine whether this process depends on the induction of oxidative stress. We demonstrate here that TNF-alpha causes a decrease in reduced glutathione (GSH) during myogenic differentiation of C(2)C(12) cells, which coincides with an elevated generation of reactive oxygen species. Supplementation of cellular GSH with N-acetyl-l-cysteine (NAC) did not reverse the inhibitory effects of TNF-alpha on troponin I promoter activation and only partially restored creatine kinase activity in TNF-alpha-treated cells. In contrast, the administration of NAC before treatment with TNF-alpha almost completely restored the formation of multinucleated myotubes. NAC decreased TNF-alpha-induced activation of NF-kappaB only marginally, indicating that the redox-sensitive component of the inhibition of myogenic differentiation by TNF-alpha occurred independently, or downstream of NF-kappaB. Our observations suggest that the inhibitory effects of TNF-alpha on myogenesis can be uncoupled in a redox-sensitive component affecting myotube formation and a redox independent component affecting myogenic protein expression.     

Tumor necrosis factor inhibits collagen and fibronectin synthesis in human dermal fibroblasts

Tumor necrosis factor (TNF) caused inhibition of collagen production by confluent cultures of human dermal fibroblasts in a dose-dependent manner. Concomitant increase of prostaglandin E2 release was observed as a result of TNF-induced cell activation. However, a blockade of the cyclooxygenase pathway of arachidonate metabolism by indomethacin did not abrogate the inhibitory effect of TNF on collagen synthesis, suggesting that this effect could be independent of prostaglandin metabolism. Gel electrophoresis of the newly synthesized macromolecules from the culture media showed that both type I and type III collagens as well as fibronectin were affected by the inhibition. Electrophoresis of cell layer-associated proteins demonstrated that the reduction in amounts of collagen and fibronectin in the medium did not result from an intracellular accumulation of these macromolecules. Production of procollagens was reduced in parallel to that of collagens, suggesting that the effect of TNF is exerted before the processing steps of procollagens. These results clearly show that TNF could play a role in modulation of matrix deposition by fibroblasts during inflammatory processes.     

Tumor necrosis factor selectively inhibits activation of human B cells by Epstein-Barr virus

We have investigated the effect of recombinant human tumor necrosis factor-alpha (rTNF-alpha) on human B cell activation and differentiation. Among several T cell-dependent and independent B cell stimulation systems tested (anti-mu, pokeweed mitogen, Epstein-Barr virus), only the activation by Epstein-Barr virus was inhibited by rTNF-alpha. rTNF-alpha inhibited in a dose-dependent manner both the proliferation and differentiation (Ig secretion) of Epstein-Barr virus-stimulated B cells when added at the beginning or within 48 hr of a 6 to 8-day culture period. Maximal suppression (80 to 95%) was found at rTNF-alpha concentrations of 10 to 50 ng/ml. Inhibition of B cell activation required the presence of significant numbers (25%) of plastic adherent macrophages within the B cell population. Suppression was not due to lysis of Epstein-Barr virus-infected B cells by rTNF-alpha-treated macrophages. As shown by double chamber experiments where macrophages and B cells were separated by a 0.45-micron membrane, macrophages elaborated factors in response to rTNF-alpha, which, alone or synergistically with rTNF-alpha, inhibited B cell activation. These factors were different from prostaglandin E2, interferon-alpha, and interleukin 1. We conclude that rTNF-alpha can dramatically modulate certain normal immune responses in vitro.     

Tumor necrosis factor release from lipopolysaccharide-stimulated human monocytes: Lipopolysaccharide tolerance in vitro

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.     

Tumor necrosis factor alpha induction in human monocytes

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein. It was found that early pretreatment with cycloheximide interferes with TNF-alpha mRNA induction by Staphylococcus aureus.     

Tumor necrosis factor in human monocyte-mediated antitumor cytotoxicity

The WEHI-164 target cells pretreated with actinomycin D can be employed in a 7-hour 51Cr release assay that exhibits exquisite susceptibility for cytotoxic monocytes without contribution by natural killer cells. The system can be used either to detect cell-mediated monocyte cytotoxicity directly or to measure cytotoxic-factor activity in cell-free supernatants. Analysis of cytotoxic factor demonstrates molecular characteristics similar to tumor necrosis factor (TNF), and polyclonal as well as monoclonal antibodies specific for TNF can readily neutralize the monocyte-generated cytotoxic factor. In the cell-mediated approach, neutralization can be achieved as well, although somewhat higher amounts of antibody are required. Hence, the WEHT-164/actinomycin D system appears to detect monocyte cytotoxicity that is mediated by TNF.     

Tumor necrosis factor inhibits K+ current expression in cultured oligodendrocytes

Summary The effects of tumor necrosis factor- (TNF-), a cytokine secreted by activated macrophages, on the electrical membrane properties of cultured adult ovine oligodendrocytes (OLGs) were investigated using the whole-cell voltage-clamp technique. Treatment with recombinant human TNF- (rhTNF) for 24 to 72 hr produces (i) process retraction in some but not all OLGs, (ii) a reduction in the resting membrane potential with no significant change in membrane capacitance or input resistance over control cells and (iii) a decrease in the expression of both the inwardly rectifying and outward K⁺ current. The magnitude of the membrane potential change as well as K⁺ current inhibition was larger in cells with retracted processes. The electrophysiological effects of rhTNF were attenuated when rhTNF was neutralized with a polyclonal anti-rhTNF antibody. The binding of rhTNF to its receptor has been reported to increase GTP binding, to increase GTPase activity of a pertussis-sensitive G protein, and to produce an elevation in intracellular cAMP in other cell types. However, pretreatment of OLGs with activated pertussis toxin failed to attenuate or mimic the effects of rhTNF. Chronic exposure of OLGs to the membrane permeant analogue of cAMP, 8-bromo-cAMP, resulted primarily in an inhibition of the inwardly rectifying K⁺ current, an effect which was less than that produced by rhTNF alone and without any of the associated rhTNF-induced morphological changes. This indicates that the effects of rhTNF cannot be entirely accounted for by an elevation in intracellular cAMP. Cycloheximide (CHX), an inhibitor of protein synthesis, mimicked the effects of rhTNF; however, the effects of rhTNF and CHX were not additive. The finding that both ionic current expression and membrane potential were reduced in cells treated with rhTNF that appeared morphologically normal suggests that abnormal ion channel expression in OLGs precedes and may contribute to eventual myelin swelling and damage.     

Tumor necrosis factor inhibits the terminal event in mesenchymal stem cell differentiation

Control of the terminal event in cellular differentiation is an important normal regulatory process, and the expression of defects in the control of this process has been implicated in the pathogenesis of cancer. To determine if tumor necrosis factor (TNF), which is an important biological response modifier, can inhibit terminal differentiation, we have studied 3T3 T mesenchymal stem cells. This experimental cell system was employed because a well-defined series of steps in differentiation has been defined and cells at each stage of differentiation can be isolated. For example, nonterminal differentiated cells can be isolated, and their transition to a terminal differentiation state can be evaluated. The most interesting results in the current studies show that TNF blocks the terminal event in mesenchymal stem cell differentiation. Inhibition of the terminal event of differentiation by TNF is reversible and is not associated with inhibition of selective or general protein synthesis. Evidence is also presented that cell clones that are defective in their ability to undergo the terminal event in differentiation secrete factor(s) that inhibit the terminal event in differentiation. These observations suggest that the inhibition of the terminal event in differentiation may be mediated via autocrine or paracrine regulatory molecules such as tumor necrosis factor.     

Tumor necrosis factor inhibits a polymorphonuclear leukocyte-dependent airway edema in guinea pigs

Intravenously administered endotoxin inhibits the polymorphonuclear leukocyte (PMN)-dependent airway edema produced in guinea pigs exposed to toluene diisocyanate (TDI). Tumor necrosis factor (TNF) is produced in vivo by peripheral blood monocytes and tissue macrophages stimulated with endotoxin and has been shown to activate PMN's and vascular endothelial cells. To determine whether the inhibition of airway edema is mediated by TNF, guinea pigs were treated with intravenous saline or 75,000 U/kg recombinant human TNF 1.5 h before exposure to air or 3 ppm TDI for 1 h. Animals were then injected intravenously with 50 mg/kg Evans blue dye as a marker of protein extravasation. Saline-treated animals exposed to TDI had a significant increase in tracheal Evans blue dye extravasation (85 +/- 6.5 micrograms dye/g trachea, mean +/- SE) compared with saline-treated animals exposed to air (31.3 +/- 2.5, P less than 0.001). The tracheal extravasation of Evans blue dye was significantly inhibited (P less than 0.05) in TDI-exposed animals treated with TNF (64.7 +/- 7.5). Neither heat-inactivated TNF (104.9 +/- 9.5) nor TNF neutralized with a monoclonal antibody against TNF (99.7 +/- 17.9) inhibited TDI-induced airway edema. In addition, treatment with 15,000 U/kg (99.9 +/- 21.3) or 150,000 U/kg (103.2 +/- 17.6) interleukin 1, a monokine also produced in response to endotoxin, did not prevent airway edema. These results suggest that TNF released in response to endotoxin mediates endotoxin's inhibition of a PMN-dependent airway edema.     

Tumor necrosis factor inhibits the proliferation of cultured capillary endothelial cells

We have examined the effect of tumor necrosis factor (TNF) on the proliferation of capillary endothelial cells derived from brain or adrenal cortex. In both cell types, TNF inhibits basal as well as basic fibroblast growth factor (bFGF)-stimulated cell proliferation. TNF induces an additional cytotoxic effect in bFGF-stimulated, but not in unstimulated, capillary endothelial cells. These results suggest that TNF could act as a negative regulator of angiogenesis in vivo and further, that TNF might induce selective cytotoxicity of capillary endothelial cells stimulated by tumor-derived bFGF. These results could explain why TNF induces hemorrhagic necrosis of certain, solid tumors.     

Tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro.

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Cytokine-mediated regulation of ovarian function. Tumor necrosis factor alpha inhibits gonadotropin-supported ovarian androgen biosynthesis

Resident ovarian macrophages have been implicated in the regulation of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, tumor necrosis factor (TNF) alpha, has been shown to attenuate the gonadotropin-dependent differentiation of the somatic ovarian (estrogen-producing) granulosa cell. This study examines the possibility that TNF alpha may also regulate the adjacent androgen-producing theca interstitial cell. The basal accumulation of androsterone (the major androgenic steroid), synthesized by whole ovarian dispersates from immature rats, remained unchanged following treatment with TNF alpha (30 ng/ml) alone. In contrast, concurrent treatment with increasing concentrations of TNF alpha (0.03-30 ng/ml), yielded dose-dependent inhibition of the human chorionic gonadotropin (1 ng/ml)-stimulated accumulation of androsterone. This reversible and immunoneutralizable effect of TNF alpha was characterized by a minimal effective dose of 0.1 ng/ml, a median inhibitory dose of 0.9 ng/ml, a maximal inhibitory effect of 90%, and a minimal time requirement of less than or equal to 48 h. Comparable results were obtained when using highly purified theca interstitial cells, thereby indicating that TNF alpha is capable of exerting a direct inhibitory effect at the level of the ovarian androgen-producing cell. TNF alpha action was not accounted for by alterations in the plated viable cell mass. Instead, treatment with TNF alpha resulted in significant inhibition of the human chorionic gonadotropin-supported accumulation of cAMP, the putative second messenger of gonadotropin hormonal action. TNF alpha action at sites distal to cAMP generation was associated with profound inhibition of the conversion of the [3H]pregnanolone (3 alpha-hydroxy,5 alpha-pregnane-20-one) and [3H]17 alpha-hydroxypregnanolone (3 alpha, 17 alpha-dihydroxy,5 alpha-pregnane-20-one) substrates to androsterone, suggesting stimulation of 20 alpha-hydroxysteroid dehydrogenase activity, inhibition of 17 alpha-hydroxylase/17:20 lyase activity, or both. Taken together, these findings indicate that TNF alpha, acting at relatively low concentrations, is capable of inhibiting gonadotropin-supported ovarian androgen biosynthesis by selectively modulating the activity of relevant key steroidogenic enzymes. As such, these observations suggest that the theca interstitial cell is a site of TNF alpha reception and action and that TNF alpha, possibly of resident ovarian macrophage origin, may partake in the regulation of ovarian androgen production, an effect due in part to inhibition of the activity of the key steroidogenic enzymes 17 alpha-hydroxylase/17:20 lyase.