Tumor Necrosis Factor-α Regulates Nicotinic Responses in Mixed Cultures of Sympathetic Neurons and Nonneuronal Cells

Abstract: It is recognized that tumor necrosis factor-α (TNF-α), a pleiotropic cytokine, influences hormone secretion and transmitter release from central neurons. To examine the role of TNF-α as a modulator of autonomic function of the PNS, we measured [³H]norepinephrine ([³H]NE) secretion evoked by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic agonist, in cultures from neonatal rat superior cervical ganglia (SCG). We found that (1) DMPP-evoked [³H]NE secretion was enhanced in SCG mixed cultures treated for 48 h with recombinant human TNF-α (rhTNF-α) plus rat interferon-γ (IFN-γ) but not in cultures treated with either cytokine alone; (2) an increase in [³H]NE secretion was also observed in mixed cultures treated with recombinant murine TNF-α (rmTNF-α) alone; and (3) the presence of nonneuronal cells or soluble factors released by them was required for the effect of these cytokines on secretion. Electrophysiologic experiments revealed an increase in nicotinic receptor current density in neurons from mixed cultures treated with rhTNF-α plus IFN-γ or with rmTNF-α when compared with control cultures. We conclude that prolonged exposure to rhTNF-α plus IFN-γ or rmTNF-α regulates nicotinic responses in SCG cultures via a soluble factor or factors secreted by nonneuronal cells.     

A follow-up to "Anti-cytokine therapy in chronic destructive arthritis" by Wim B van den Berg

In recent years, the effectiveness of anti-TNF therapy in treating rheumatoid arthritis (RA) has become apparent. While trials of IL-1 receptor antagonist in RA have been encouraging, it clearly is more difficult to target two molecules (IL-1 α and β) than one (TNF-α). In his review article, Professor Wim van den Berg argues that both TNF-α and IL-1 must be blocked in RA and that although TNF is clearly a potent inflammatory molecule, the dominant cytokine in the subsequent degradation of the joint tissue is IL-1. This commentary discusses his hypothesis in light of animal studies and the limitations of the conclusions that can be drawn from them. More broadly, it discusses the biology of TNF-α and IL-1 and suggests explanations of why TNF-α is a pivotal cytokine in this disease.     

Effect of GM-CSF on cytokine induction by soluble β-glucan SCG in vitro in β-glucan-treated mice

SCG is a 6-branched 1,3-β- d -glucan, which are major cell wall structural components in fungi. Leukocytes from DBA/1 and DBA/2 mice are highly sensitive to SCG, producing cytokines such as GM-CSF, IFN-γ, TNF-α and IL-12p70, but not IL-6. GM-CSF plays a key biological role in this activity. In the present study, we examined the effect of giving i.p. SCG to DBA/2 mice on cytokine production in vitro . SCG was given i.p. to DBA/2 mice on day 0. Splenocytes were prepared on day 7 and cultured in the presence of SCG in vitro . The levels of cytokine production induced by SCG in vitro were lower in the cells from SCG-treated mice than in control mice. Expression of the β-glucan receptor, dectin-1, in SCG-treated mice was comparable with that shown in control mice. However, the consumption of exogenously added rmGM-CSF in vitro was observed in SCG-treated mice. The addition of a large amount of rmGM-CSF to the culture medium resulted in larger amounts of TNF-α and IL-6 in SCG-treated mice than in normal mice. These results suggested that GM-CSF was closely related with the reactivity of β-glucan. Giving SCG increased the number of macrophages and granulocytes in the spleen. These results suggested that in SCG-treated mice, a change of cell population would be related to modulation of the profile of cytokine production induced by SCG in vitro .     

Immunohistochemical localization and possible physiological roles of tumor necrosis factor-α in mouse cumulus-oocyte complexes

Tumor necrosis factor-α (TNF-α), a cytokine which is produced by activated macrophages, has been shown to participate in the regulation of ovarian functions. In the course of our investigation on the mechanism of maturation, fertilization and degeneration of mouse oocytes, immunoreactivity to TNF-α was found in the cytoplasm of the cells surrounding the maturing oocytes and of granulosa cells facing the antral cavity. Immunoblot analysis with the specific antibody to TNF-α identified the 17 kDa Mr band in the extract of cumulusoocyte complexes. Various concentrations of TNF-α (mouse, recombinant) and anti TNF-α antiserum (polyclonal rabbit anti-mouse recombinant TNF-α) were then used to determine their effect on the germinal vesicle breakdown (GVBD), polar body extrusion, fertilization and fragmentation of mouse oocytes/eggs. TNF-α at concentrations of 10 ng/mL or less and anti-TNF-α antiserum at concentrations of 10% or less, had no effect on the spontaneous GVBD and polar body extrusion of mouse oocytes in culture. Mouse follicular oocytes cultured for more than 72 h in modified Krebs-Ringer solution in vitro undergo spontaneous fragmentation, which is a degenerative change to form 'blastomeres' with or without nuclear fragments or chromatin. Ghost-like blastomeres were also identified in the space among fragmented 'blastomeres'. The spontaneous fragmentation of mouse follicular oocytes was suppressed in the presence of TNF-α at concentrations of 1 ng/mL or greater. Anti-TNF-α antiserum (1%) accelerated the induction of fragmentation of oocytes cultured in vitro . The addition of anti TNF-α antiserum (10%) to the culture medium did not influence the fertilization rates of the eggs surrounded by the expanded cumulus. These results appear to indicate that the process of degeneration of mouse oocytes/eggs is modulated by TNF-α accumulated in the expanded cumulus during oocyte maturation.     

Cellular Localization and Temporal Elevation of Tumor Necrosis Factor-α, Interleukin-1α, and Transforming Growth Factor-β1 mRNA in Hippocampal Injury Response Induced by Trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6, and transforming growth factor-β (TGF-β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17-day-old mouse hippocampus during trimethyltin-induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF-α, IL-1α, IL-1β, and IL-6 mRNA were seen. TGF-β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF-α and IL-1α were localized to the microglia, whereas TGF-β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule-1, EB-22, Mac-1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon-γ mRNA. These data suggest that pro-inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.     

Tumor necrosis factor-alpha (TNF-alpha) regulates Toll-like receptor 2 (TLR2) expression in microglia

Microglia represent one effector arm of CNS innate immunity as evident by their role in pathogen recognition. We previously reported that exposure of microglia to Staphylococcus aureus ( S. aureus), a prevalent CNS pathogen, led to elevated Toll-like receptor 2 (TLR2) expression, a pattern recognition receptor capable of recognizing conserved structural motifs associated with gram-positive bacteria such as S. aureus . In this study, we demonstrate that the proinflammatory cytokine tumor necrosis factor-α (TNF-α) enhances TLR2 expression in microglia, whereas interleukin-1β has no significant effect. To determine the downstream signaling events responsible for elevated microglial TLR2 expression in response to TNF-α, a series of signal transduction inhibitors were employed. Treatment with caffeic acid phenethyl ester, an inhibitor of redox-mediated nuclear factor-kappa B activation, significantly attenuated TNF-α-induced TLR2 expression. Similar results were observed with the IKK-2 and IκB-α inhibitors SC-514 and BAY 11-7082, respectively. In contrast, no significant alterations in TLR2 expression were observed with protein kinase C or p38 mitogen-activated protein kinase inhibitors. A definitive role for TNF-α was demonstrated by the inability of S. aureus to augment TLR2 expression in microglia isolated from TNF-α knockout mice. In addition, TLR2 expression was significantly attenuated in brain abscesses of TNF-α knockout mice. Collectively, these results indicate that in response to S. aureus , TNF-α acts in an autocrine/paracrine manner to enhance TLR2 expression in microglia and that this effect is mediated, in part, by activation of the nuclear factor-kappa B pathway.     

Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

Muscle precursor cells isolated from aged rats exhibit an increased tumor necrosis factor-α response

Improving muscle precursor cell (MPC, muscle-specific stem cells) function during aging has been implicated as a key therapeutic target for improving age-related skeletal muscle loss. MPC dysfunction during aging can be attributed to both the aging MPC population and the changing environment in skeletal muscle. Previous reports have identified elevated levels of tumor necrosis factor-α (TNF-α) in aging, both circulating and locally in skeletal muscle. The purpose of the present study was to determine if age-related differences exist between TNF-α-induced nuclear factor-kappa B (NF-κB) activation and expression of apoptotic gene targets. MPCs isolated from 32-month-old animals exhibited an increased NF-κB activation in response to 1, 5, and 20 ng mL⁻¹ TNF-α, compared to MPCs isolated from 3-month-old animals. No age differences were observed in the rapid canonical signaling events leading to NF-κB activation or in the increase in mRNA levels for TNF receptor 1, TNF receptor 2, TNF receptor-associated factor 2 (TRAF2), or Fas (CD95) observed after 2 h of TNF-α stimulation. Interestingly, mRNA levels for TRAF2 and the cell death-inducing receptor, Fas (CD95), were persistently upregulated in response to 24 h TNF-α treatment in MPCs isolated from 32-month-old animals, compared to 3-month-old animals. Our data indicate that age-related differences may exist in the regulatory mechanisms responsible for NF-κB inactivation, which may have an effect on TNF-α-induced apoptotic signaling. These findings improve our understanding of the interaction between aged MPCs and the changing environment associated with age, which is critical for the development of potential clinical interventions aimed at improving MPC function with age.     

Effect of tumor necrosis factor-α on intracellular Staphylococcus aureus in vascular endothelial cells

Although tumor necrosis factor-α (TNF-α) is an important host factor against intracellular bacteria, little is known about the effect of TNF-α on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-α influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-α, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10⁶ CFU well⁻¹) into HUVECs for 1 h. TNF-α (1 ng mL⁻¹) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-α plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-α preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-α helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-α preferentially induces apoptosis of endothelial cells infected by S. aureus .     

In vitro neutralization of tumor necrosis factor-α during Chlamydia pneumoniae infection impairs dendritic cells maturation/function and increases chlamydial progeny

Dendritic cells (DCs) produce tumor necrosis factor (TNF)-α upon infection and contribute in various ways to defense against pathogenic agents. Several biological agents have been designed to inhibit TNF-α activity. However, the use of these inhibitors has been associated with an increased rate of certain opportunistic infections. To study the effect of TNF-α inhibition, human monocyte-derived DCs were infected with Chlamydia pneumoniae . TNF-α was neutralized by adalimumab, a human anti-TNF-α monoclonal antibody. Chlamydiae induced the maturation of DC as determined by flow cytometry and quantitative real-time PCR. However, DC maturation was impaired in the presence of adalimumab. Moreover, neutralization of TNF-α resulted in a significant increase of infectious progeny, 16S rRNA gene copy number and development of larger inclusions consisting of different stages of chlamydial development. Additionally, chlamydial infection induced secretion of cytokines/chemokines, which were downregulated by adalimumab treatment. Our data reveal an indirect effect on maturation of DC by C. pneumoniae and that maturation is crucial for the restriction of chlamydial development. The results also demonstrate an increase in infectious progeny after TNF-α inhibition, suggesting a contribution of TNF-α produced by DCs to chlamydial growth arrest. These data suggest a possible mechanism by which TNF-α inhibition enhances the risk of intracellular infections.     

Cytokine-Regulated Expression of Platelet-Derived Growth Factor Gene and Protein in Cultured Human Astrocytes

Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β₁ (TGF-β₁) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β₁ in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β₁, TNF-α, TNF-α/TGF-β₁, or IL-1β/TGF-β₁, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β₁, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Effects of murine lysozyme on lipopolysaccharide-induced biological activities

Abstract We have demonstrated that egg-white lysozyme (EW-LZM) bound to lipopolysaccharide (LPS), reduced the lethal toxicity and the biological activity of LPS. In this study, the interaction of LPS with murine lysozyme (M-LZM) and the modulation of biological activities were investigated. M-LZM was prepared from the culture supernatant of the murine macrophage cell line RAW264.7 by ion-exchange and gel filtration chromatographies and dialysis. Two types of M-LZM, murine M lysozyme (MM-LZM) and murine P lysozyme (MP-LZM), were purified from the supernatant. The enzymatic activities of both MM-LZM and MP-LZM were inhibited by LPS and their effects were affected by the temperature and the ionic strength. TNF-α production from RAW264.7 by LPS was inhibited by mixing with MM-LZM and MP-LZM. MP-LZM inhibited TNF-α production stronger than MM-LZM. Considering these facts, we suggested that M-LZM, like EW-LZM, make a complex with LPS to reduce the toxicity of LPS together with inhibiting the enzymatic activity.