Regulation of tumor necrosis factor-α expression by CD40 ligation in BV-2 microglial cells

Ligation of CD40 has been shown to induce/stimulate the expression of tumor necrosis factor-alpha (TNF-alpha) in microglial cells. This study delineates the mechanism by which CD40 ligation regulates the expression of TNF-alpha in BV-2 microglial cells. There was very little induction of TNF-alpha by ligation of CD40 alone by either cross-linking antibodies against CD40 or recombinant CD40 ligand (CD154). The absence of any increase in TNF-alpha production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggest that signal transduced by the ligation of CD40 alone is not sufficient for strong induction of TNF-alpha. However, CD40 ligation markedly induced the production of TNF-alpha as well as the expression of TNF-alpha mRNA in interferon-gamma (IFN-gamma)-stimulated BV-2 glial cells. Ligation of CD40 in CD40-overexpressed cells markedly enhanced the expression of TNF-alpha in the presence of IFN-gamma. To understand the mechanism of CD40 ligation-mediated induction/stimulation of TNF-alpha, we investigated the role of nuclear factor-kappaB (NF-kappaB) and C/EBPbeta. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone in the presence or absence of CD40 overexpression induced the activation of only NF-kappaB and not that of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of TNF-alpha and that the activation of C/EBPbeta is also necessary for strong induction of TNF-alpha. Consistently, a dominant-negative mutant of p65 (Delta(p65)) and that of C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of TNF-alpha in BV-2 microglial cells stimulated with the combination of IFN-gamma and CD40 ligand. Taken together, these studies suggest that activation of both NF-kappaB and C/EBPbeta is important for strong induction of TNF-alpha and that CD40 ligation regulates the expression of TNF-alpha by modulating the activation of only NF-kappaB but not that of C/EBPbeta.     

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 .     

TNF-α reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor α (TNF-α). Therefore, this work was designed to explore the effect of TNF-α on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-α caused a marked reduction in the number of intracellular S. epidermidis , suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-κB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-κB inhibitors, SN50 or BAY 11–7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis . These results suggest that activation of the signaling pathway initiated by TNF-α could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-κB was shown not to be important for the internalization process of S. epidermidis .     

Down-regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type-1 receptor and nuclear factor-κB activation

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.     

Increased susceptibility to tumor necrosis factor-α in butyric acid-induced apoptosis is caused by downregulation of cFLIP expression in Jurkat T cells

Butyric acid is one of the major extracellular metabolites of periodontopathic Gram-negative bacteria. We previously demonstrated that butyric acid induced apoptosis in human T cells. In the present study, we examined the interaction between butyric acid and TNF-α in Jurkat T-cell apoptosis. Simultaneous treatment with TNF-α enhanced butyric acid-induced apoptosis by promoting caspase activity more than was achieved by either reagent alone. We examined which genes were associated with the increased susceptibility to TNF-α caused by butyric acid, and revealed that expression of cFLIP decreased with increased concentrations of butyric acid. Furthermore, exogenous expression of cFLIP protein suppressed the enhancing effect by TNF-α in the apoptosis. These results suggest that butyric acid downregulates cFLIP expression and increases the susceptibility to TNF-α by activating caspases via the death receptor signal.     

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.     

Dual role of the caspase enzymes in satellite cells from aged and young subjects

Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage.     

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.     

Peptidoglycan and teichoic acid from Staphylococcus epidermidis stimulate human monocytes to release tumour necrosis factor-α, interleukin-1β and interleukin-6

Abstract Cytokines play a major role in the pathophysiology of septic shock. In this study, human periferal blood monocytes were stimulated with peptidoglycan and teichoic acid, purified from a strain of Staphylococcus epidermidis . Polymyxin B (PM-B) was added to avoid the effects of possible contamination with endotoxin. Tumour necrosis factor-α (TNF), interleukin-1β (IL-1), and interleukin-6 (IL-6) in the supernates were measured by enzyme-linked immunosorbent assays. Peptidoglycan and teichoic acid induced TNF, IL-1, and IL-6 in a concentration-dependent manner. Teichoic acid was a weaker inducer than peptidoglycan, especially for IL-1. Lipopolysaccharide from an E. coli strain was used as a control, being 100–1000 times more potent than peptidoglycan and teichoic acid.     

Biological characteristics of muscle-derived satellite cells isolated from rats at different postnatal days

This study investigated the in vitro growth characteristics and differential potential of muscle-derived satellite cells (MDSCs) derived from rats at different postnatal (P) stages, in order to expand the range of source material for tissue engineering. Rat MDSCs were isolated from P5, P10, P15, P21 and P42 rat skeletal muscles using double enzyme digestion and differential adherent culture. Neurogenic, osteogenic and myogenic induction media were used to induce directed differentiation. Differentiated nerve cells, osteoblasts and myotubes were identified by their morphology and immunohistochemical staining. Most cells transformed into spindle-shaped mononuclear cells after 48 h and proliferated rapidly. MDSCs were difficult to isolate from P42 rats. After neurogenesis, four groups MDSCs formed neuron-specific enolase positive polygonal-shaped dendritic cells. After osteogenesis, P5, P10, P15 and P21 MDSCs formed Alizarin red- and osteocalcin-positive bone nodules. After myogenesis, myotubes were formed and were fast muscle myosin-positive. MDSCs derived from P5, P10, P15 and P21 rat skeletal muscle are easy to isolate, culture and amplify in vitro, which increases the range of source material available for tissue engineering.     

Attempts to characterize the mechanisms involved in the growth inhibition of Mycobacterium microti in interferon-γ or tumor necrosis factor-α activated J774A.1 cells

Abstract The growth of Mycobacterium microti was inhibited within J774A. 1 macrophage cells activated with either interferon-γ or tumor necrosis factor-α. Activation with interferon-γ or tumor necrosis factor-α alone did not stimulate the production of nitrite in J774A. 1 cells. Interferon-γ but not tumor necrosis factor-a increased the production of hydrogen peroxide in a concentration dependent manner but scavengers of reactive oxygen species did not influence the growth inhibiting effect of interferon-γ within J774A.1 cells. Both interferon-γ and tumor necrosis factor-α enhanced the fusion of M. microti containing phagosomes with lysosomes and the ultimate degradation of bacteria. Our results showed that growth inhibition of M. microti within interferon-γ or tumor necrosis factor-a stimulated J774A. 1 cells was independent of reactive oxygen intermediate and reactive nitrogen intermediate production.     

Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

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Giardia lamblia: Interleukin 6 and tumor necrosis factor-alpha release from mast cells induced through an Ig-independent pathway

Giardia lamblia is a common cause of both acute and chronic diarrheal disease in humans worldwide. It has been shown that mast cells, IL-6 and TNF-α are substantially involved in the early control of G. lamblia infection in mice. However, no studies have yet been reported concerning the interaction between mast cell and Giardia, as well as the mast cells mediators generated in response to Giardia infection. In this study we demonstrated the direct activation of mast cells by G. lamblia live trophozoites or trophozoite-derived antigens followed by an increase in tryptase expression and a significant release of the preformed mediator histamine. In addition, parasite derived antigens increased TNF-α and de novo synthesized cytokine IL-6, at the mRNA and protein level. These results strongly suggest that mast cells might be an important source not only of IL-6 but also of TNF-α during Giardia infection, playing an important role in the outcome of the infection.     

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2-transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24-72 h in medium without BS containing either vehicle, TNF-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 caused a significant decrease in the number of wild-type cells. This decrease was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with TNF-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-induced cell death was significantly prevented in culture with caspase-3 inhibitor (10(-8) M). Nitric oxide (NO) synthase activity in wild-type cells was significantly increased by addition of calcium chloride (10 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB mRNAs was significantly increased in transfectants as compared with that of wild-type cells. Smad 3 or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF-alpha or TGF-beta1 in kidney NRK52E cells.     

CD8<Superscript>+</Superscript> T lymphocytes isolated from renal cancer patients recognize tumour cells through an HLA- and TCR/CD3-independent pathway

PURPOSE: The aim of this study was to characterize the immune response of patients affected by renal cell carcinoma (RCC). METHODS: Long-term RCC lines were established by retroviral-mediated transfer of the large T-antigen of SV40 into fresh carcinoma cells. Reactive T cell effectors were generated by iterative stimulations of patients' PBMC with autologous tumour cells. RESULTS: This protocol led to the induction of CD8(+) T cell clones reactive against the autologous tumour, but not against NK-sensitive cell lines. However, some of these effectors recognize normal renal cells, allogeneic renal carcinoma cell lines and colon and non-small cell lung carcinomas but not melanomas and lymphoblastoid lines, without evidence of shared classical HLA class I (HLA-I) molecules. Further characterization performed on the CD8(+) TCR alpha/beta(+) clone, CTL30, demonstrated that neither expression of CD1, HLA-Ia nor HLA-Ib, correlated with the T cells' recognition. Moreover, beta2m expression by target cells was not required to achieve interaction of tumour-effector cells. In agreement with this observation, the lytic activity of CTL30 was not inhibited by anti-HLA-I Ab, and antigen expression was not affected by inhibitors of antigen processing. Lytic activity of CTL30, while partially inhibited by anti-NKG2D, could not be abolished by anti-CD3 Abs. Moreover, growth and expansion of CTL30 was sustained only by T cell interaction with antigen-expressing tumour cells; unspecific mitogenic stimuli, such as anti-CD3 and PHA, did not allow T cell expansion. These results demonstrated the existence of an alpha/beta T cell population, recognizing epithelial tumour cells through an HLA-unrestricted, CD3-independent mechanism.     

Effects of an oriental herbal medicine, “Saiboku-to”, and its constituent herbs on Compound 48/80-induced histamine release from peritoneal mast cells in rats

Effects of a traditional oriental herbal medicine, "Saiboku-to" and its constituent herbs on Compound 48/80-induced histamine release from peritoneal mast cells in rats were investigated. Saiboku-to inhibited Compound 48/80-induced degranulation of and histamine release from the mast cells, suggesting that Saiboku-to not only possesses anti-histamine release effect from mast cells, but also contains active herbs with this effect. Significant inhibitions were found in 4 of 10 constituent herbs of Saiboku-to: Magnoliae Cortex, Perillae Herba, Bupleuri Radix and Hoelen. In the dose-response curves of the four herbs, the logarithmic linearity was observed for each herb, and 50% inhibitory concentration, the IC50 values, were calculated to be 56.8 microg/ml for Magnoliae Cortex, 175.8 microl/ml for Perillae Herba, 356.6 microg/ml for Bupleuri Radix, and 595.8 microg/ml for Hoelen. One mg/ml of Saiboku-to showing 75% inhibition of Compound 48/80-induced histamine release level from mast cells contains 88.5 microg of Magnoliae Cortex (it was estimated from the dose-response curve that this dose inhibits 62.68% of the Compound 48/80-induced histamine release level), 58.8 microg of Perillae Herba (21% inhibition), 205.9 microg of Bupleuri Radix (35.24% inhibition), and 147.1 microg of Hoelen (11.15% inhibition). From these results, it is suggested that the anti-histamine release effect of Saiboku-to, which contains 10 herbs, may be due mainly to the effect of Magnoliae Cortex and the synergism of the 3 other herbs.