In vitro and in vivo inhibition of LPS-induced tumor necrosis factor-alpha production by dimeric gallotannin analogues.

Designed dimeric gallotannin analogues featuring two tetragalloylglucopyranose cores connected by various hydrocarbon linkers inhibit tumor necrosis factor-alpha secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells by up to 53% (5-24 microM concentration range) compared to control. Comparable suppression of tumor necrosis factor-alpha levels (approximately 50% vs control) was observed in the plasma of rats co-treated with lipopolysaccharide and specific tannin analogues selected for their lack of interleukin 1-beta stimulating activity.     

Probiotic genomic DNA reduces the production of pro-inflammatory cytokine tumor necrosis factor-alpha

The effect of Lactobacillus plantarum genomic DNA on lipopolysaccharide (LPS)-induced mitogen-activated protein kinase (MAPK) activation, nuclear factor-kappa B activation, and the expressions of tumor necrosis factor-alpha, interleukin-1 receptor-associated kinase M, and the pattern recognition receptor were examined. Pretreatment of p-gDNA inhibited the phosphorylation of MAPKs and nuclear factor-kappa B, and also inhibited LPS-induced TNF-α production in response to subsequent LPS stimulation. L.?plantarum genomic DNA-mediated inhibition of signaling pathway and tumor necrosis factor-alpha was accompanied by the suppression of toll-like receptor (TLR) 2, TLR4, and TLR9 and the induction of interleukin-1 receptor-associated kinase M, a negative regulator of TLR. This study can extend our understanding of the biological function of probiotic genomic DNA as an anti-inflammatory agent.     

Lipopolysaccharide-induced inhibition of scavenger receptor expression in human monocyte-macrophages is mediated through tumor necrosis factor-alpha.

The exposure of mononuclear cells to LPS results in a variety of cellular alterations including changes in the expression of various membrane receptors. In human monocyte-macrophages the development of the scavenger receptor, which mediates the uptake and internalization of chemically modified proteins, was suppressed by 100 ng/ml of LPS, concomitant with a reduction in receptor mRNA. Removal of LPS from the media resulted in a rapid increase in scavenger-receptor activity and mRNA that was further enhanced by macrophage-CSF and granulocyte/macrophage-CSF. However, neither macrophage-CSF nor granulocyte/macrophage-CSF could overcome the suppression of scavenger-receptor activity in the presence of LPS. The LPS-induced suppression of the scavenger receptor could be overcome by the co-addition of neutralizing antibody to TNF-alpha. TNF-alpha added to human monocyte-macrophages in the absence of LPS suppressed scavenger receptor activity to the same extent as did LPS. In contrast, the co-addition of LPS and neutralizing antibodies to either IFN-gamma or to IL-1 beta did not overcome the inhibitory effects of LPS on scavenger receptor activity. We conclude that the LPS-induced suppression of the scavenger receptor is mediated primarily through TNF-alpha.     

Serotonin inhibition of tumor necrosis factor-alpha synthesis by human monocytes

Serotonin inhibited in a concentration dependent way (10(-3) M to 10(-10) M) the LPS induced Tumor Necrosis Factor-alpha synthesis both, when added to the monocyte cultures from the beginning and when added together with the activating stimulus 8 hours before the end of the culture. The inhibitory effect was specifically blocked by the 5-HT1 and 5-HT2 serotonin antagonist methysergide and the 5-HT2 receptor antagonist ketanserin. This indicates that only the 5-HT2 receptor family (5-HT2 or 5-HT1C) may be involved in the inhibitory effect. Serotonin seems to play an important immunomodulatory role in macrophage functions.     

Interleukin-10 inhibits tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-alpha production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-alpha production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-kappaB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-alpha production via reduced MyD88 expression.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxifylline

Tumor necrosis factor-alpha (TNF), a mononuclear phagocyte (MO)-derived peptide, is increasingly being recognized for its pleomorphic immunologic effects. A number of investigations have demonstrated that lipopolysaccharide (LPS) can induce TNF synthesis, yet mechanisms that regulate TNF expression at the cellular and molecular levels have not been fully elucidated. In this study, we present data demonstrating pentoxifylline, a methylxanthine, is efficacious in suppressing LPS-induced MO-derived TNF at the level of both TNF mRNA accumulation and TNF supernatant bioactivity. Pentoxifylline, at a dose of 1 x 10(-5)M, suppressed the production of both biologically active TNF and TNF mRNA expression by more than 50%. Furthermore, additional methylxanthines and dibutyryl cAMP have similar effects on TNF expression. These data support the mechanism for this suppressive effect is via the generation of intracellular cAMP.     

Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.     

Insulin up-regulates tumor necrosis factor-alpha production in macrophages through an extracellular-regulated kinase-dependent pathway.

Hyperinsulinemia has recently been reported as a risk factor for atherosclerotic diseases such as coronary heart disease; however, the effect of insulin on the development of atherosclerosis is not well understood. Here we have investigated the direct effect of insulin on macrophages, which are known to be important in the atherosclerotic process. We treated THP-1 macrophages with insulin (10(-7) m) and examined the gene expression using nucleic acid array systems. The results of array analysis showed that insulin stimulated gene expression of tumor necrosis factor-alpha (TNF-alpha) the most among all genes in the analysis. In addition, insulin administration to macrophages enhanced both mRNA expression and protein secretion of TNF-alpha in a dose-dependent manner. To determine the signaling pathway involved in this TNF-alpha response to insulin, we pretreated the cells with three distinct protein kinase inhibitors: wortmannin, PD98059, and SB203580. Only PD98059, which inhibits extracellular signal-regulated kinases, suppressed insulin-induced production of TNF-alpha mRNA and protein in THP-1 macrophages. These observations indicate that insulin stimulates TNF-alpha production in macrophages by regulating the expression of TNF-alpha mRNA and that the extracellular signal-regulated kinase signaling pathway may have a critical role in stimulating the production of TNF-alpha in response to insulin in macrophages.     

Differential priming for endotoxin-induced circulating cytokine production by tumor necrosis factor-alpha and interleukin 1 beta

In unprimed mice, a single injection of a non-lethal dose of lipopolysaccharide (LPS) produced a rise in tumor necrosis factor (TNF) and interleukin 6 (IL 6) activities. Peak serum concentrations were attained, respectively, 1.5 hr and 2.5 hr after the challenge. Pretreatment with recombinant human TNF-alpha (rHuTNF) had a priming effect for enhanced production of both serum cytokines without any change in kinetics. The enhancement was more pronounced in the TNF (15-fold) than in the IL 6 (4-fold) response. Recombinant murine TNF caused a comparable increase in LPS-induced cytokine release. In contrast, comparable pretreatment with another macrophage-derived cytokine, recombinant human interleukin 1 beta (HuIL1-beta), revealed a negative effect on LPS-induced TNF release whereas IL 6 in the blood reached levels similar to those found after priming with rTNF. Moreover, when administered in combination with rHuTNF, rHuIL1-beta inhibited the priming effect on TNF autocrine production.     

Inhibition of lipopolysaccharide-induced interleukin-1beta and tumor necrosis factor-alpha production by menthone through nuclear factor-kappaB signaling pathway in HaCat cells

Menthone, the Chinese old remedy extracted from genus Mentha, has been widely used as a cooling agent, a counterirritant for pain relief, and for the treatment of pruritus. However, its detail mechanisms for interfering inflammatory reaction remain unknown. In this study, we found that menthone can suppress the lipopolysaccharide (LPS)-induced proinflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as nuclear factor kappaB (NF-kappaB) activity induced by LPS and other inflammatory agents, including 12-O-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid, and ceramide. Furthermore, our data also demonstrated that the translocation of NF-kappaB activated by LPS into the nucleus was suppressed by menthone, and I-kappaB and beta-transducin repeat containing protein (beta-TrCP) were both involved in this suppression. To sum up, this study has provided molecular evidence for menthone effect on the LPS-induced cytokine production, NF-kappaB activation, and the involvement of I-kappaB and beta-TrCP.     

Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-alpha

Uncoupling protein-2 (UCP-2) is amitochondrial protein expressed in adipocytes and has recently beeninvolved in the control of energy dissipation. Because obesity ischaracterized by an imbalance between energy intake and expenditure andby an enhanced adipocyte-derived secretion of tumor necrosis factor-(TNF-), we asked whether TNF- could directly influence UCP-2expression in adipocytes. Experiments performed in differentiated3T3F442A preadipocytes showed that TNF- (10 ng/ml) induced areduction of UCP-2 trancripts, assessed by Northern blot analysis. Asignificant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF- stimulation of the cells. The characterizationof the mechanisms responsible for the TNF- effect on UCP-2expression demonstrates an involvement of the TNF--induced inducible(i) nitric oxide synthase (NOS) expression. Cell treatment with the NOSinhibitor N^G-nitro-L-arginine methylester (L-NAME; 1 mmol/l) significantly diminished theTNF--mediated sustained downregulation of UCP-2 expression, whereascell treatment with a nitric oxide (NO) donor (10³ mol/lS-nitroso-L-glutathione) mimicked the TNF-effect on UCP-2 expression. Moreover, Western blot analysis clearlyshowed that TNF- alone induces the expression of iNOS after12-24 h treatment of differentiated 3T3F442A cells. Theseexperiments demonstrate that TNF- directly downregulates UCP-2expression via NO-dependent pathways that involve the induction of iNOS expression.

     

Inhibition of muscle insulin-like growth factor I expression by tumor necrosis factor-alpha

The role of TNF-alpha in muscle catabolism is well established, but little is known about the mechanisms of its catabolic action. One possibility could be that TNF-alpha impairs the production of local growth factors like IGF-I. The aim of this study was to investigate whether TNF-alpha can directly inhibit IGF-I gene and protein expression in muscle. First, we investigated whether the acute inflammation induced by endotoxin injection changes IGF-I and TNF-alpha mRNA in rat tibialis anterior muscle. Endotoxin rapidly increased TNF-alpha mRNA (7-fold at 1 h, P < 0.001) and later decreased IGF-I mRNA (-73% at 12 h, P < 0.001). Furthermore, in a model of C2C12 myotubes, TNF-alpha strongly inhibited IGF-I mRNA and protein (-73 and -47% after 72 h, P < 0.001 and P < 0.01, respectively). Other proinflammatory cytokines failed to inhibit IGF-I mRNA. The effect of TNF-alpha on IGF-I mRNA was not mediated by nitric oxide, and the activation of NF-kappaB was insufficient to inhibit IGF-I expression. Taken together, our data suggest that TNF-alpha induced in muscle after LPS injection can locally inhibit IGF-I expression. The inhibition of muscle IGF-I production could contribute to the catabolic effect of TNF-alpha.     

Short-term treatment of RAW264.7 macrophages with adiponectin increases tumor necrosis factor-alpha (TNF-alpha) expression via ERK1/2 activation and Egr-1 expression: role of TNF-alpha in adiponectin-stimulated interleukin-10 production

Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-alpha expression in RAW264.7 macrophages; this TNF-alpha then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-alpha mRNA accumulation were associated with increased TNF-alpha promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFkappaB; mutation of either the Egr-1 or NFkappaB binding sites in the TNF-alpha promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IkappaB super-repressor prevented gAcrp-stimulated TNF-alpha promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-alpha promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-alpha production by macrophages via ERK1/2-->Egr-1 and NFkappaB-dependent mechanisms; these increases in TNF-alpha in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.