The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Regulation of adiponectin production by insulin: interactions with tumor necrosis factor-α and interleukin-6

Obesity is often associated with insulin resistance, low-grade systemic inflammation, and reduced plasma adiponectin. Inflammation is also increased in adipose tissue, but it is not clear whether the reductions of adiponectin levels are related to dysregulation of insulin activity and/or increased proinflammatory mediators. In this study, we investigated the interactions of insulin, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in the regulation of adiponectin production using in vivo and in vitro approaches. Plasma adiponectin and parameters of insulin resistance and inflammation were assessed in a cohort of lean and obese insulin-resistant subjects. In addition, the effect of insulin was examined in vivo using the hyperinsulinemic-euglycemic clamp, and in adipose tissue (AT) cultures. Compared with lean subjects, the levels of total adiponectin, and especially the high-molecular-weight (HMW) isomer, were abnormally low in obese insulin-resistant subjects. The hyperinsulinemic clamp data confirmed the insulin-resistant state in the obese patients and showed that insulin infusion significantly increased the plasma adiponectin in lean but not obese subjects (P < 0.01). Similarly, insulin increased total adiponectin release from AT explants of lean and not obese subjects. Moreover, expression and secretion of TNF-α and IL-6 increased significantly in AT of obese subjects and were negatively associated with expression and secretion of adiponectin. In 3T3-L1 and human adipocyte cultures, insulin strongly enhanced adiponectin expression (2-fold) and secretion (3-fold). TNF-α, and not IL-6, strongly opposed the stimulatory effects of insulin. Intriguingly, the inhibitory effect of TNF-α was especially directed toward the HMW isomer of adiponectin. In conclusion, these studies show that insulin upregulates adiponectin expression and release, and that TNF-α opposes the stimulatory effects of insulin. A combination of insulin resistance and increased TNF-α production could explain the decline of adiponectin levels and alterations of isomer composition in plasma of obese insulin-resistant subjects.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor–stroma interaction

Background

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.

Methods

EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.

Results

The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.

Conclusion and general significance

The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Trans-10, cis-12-conjugated linoleic acid attenuates tumor necrosis factor-α production by lipopolysaccharide-stimulated porcine peripheral blood mononuclear cells through induction of interleukin-10

Conjugated linoleic acid (CLA) can stimulate or inhibit immune cell function, and among CLA isomers, trans-10, cis-12 (t10c12)-CLA was shown to participate in the modulation of pro- or anti-inflammatory cytokine secretion. The objective of this study was to examine the effect of t10c12-CLA on tumor necrosis factor (TNF)-α production by lipopolysaccharide (LPS)-stimulated porcine peripheral blood mononuclear cells (PBMCs). In addition, we determined whether these effects were associated with the induction of interleukin (IL)-10. Treatment of LPS-unstimulated porcine PBMCs with t10c12-CLA increased both TNF-α expression and IL-10 production. However, treatment of LPS-stimulated porcine PBMCs with t10c12-CLA suppressed TNF-α production and increased the levels of IL-10. Furthermore, treatment of LPS-stimulated porcine PBMCs with IL-10 suppressed the production of TNF-α. The effects of t10c12-CLA on TNF-α expression by both LPS-naïve and LPS-stimulated PBMCs were inhibited by IL-10 treatment. The suppressive effects of t10c12-CLA on TNF-α production by LPS-stimulated porcine PBMCs were inhibited by an anti-IL-10 polyclonal antibody. These findings suggest that t10c12-CLA has an immunostimulatory effect on porcine PBMCs mediated via the up-regulation of TNF-α production, and an anti-inflammatory effect in LPS-stimulated PBMCs mediated via the down-regulation of TNF-α production, and that both is likely to be associated with the induction of IL-10.     

Gender difference in tumor necrosis factor-α production in human neutrophils stimulated by lipopolysaccharide and interferon-γ

The gender difference in tumor necrosis factor-α (TNF-α) production in human neutrophils stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was explored by using peripheral blood neutrophils from young men and women. As compared with female neutrophils, male neutrophils released greater amounts of TNF-α, and exhibited stronger activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase in response to LPS stimulation. LPS-induced TNF-α production was markedly enhanced by pretreatment of cells with IFN-γ, and IFN-γ-mediated priming in male neutrophils was significantly greater than that in female neutrophils. Male neutrophils showed higher expression of TLR4, but not IFN-γ receptors, than female neutrophils, and its expression was increased by stimulation with IFN-γ or IFN-γ plus LPS. These findings indicate that male neutrophils show higher responsiveness to stimulation with LPS and IFN-γ than female neutrophils, and suggest that the gender difference in neutrophil responsiveness to LPS and IFN-γ is partly responsible for that in the outcome of sepsis, in which premenopausal women show a favorable prognosis as compared with men.     

Characterisation of the prostaglandin E2-ethanolamide suppression of tumour necrosis factor-α production in human monocytic cells

Background and purpose

Prostaglandin ethanolamides or prostamides are naturally occurring neutral lipid derivatives of prostaglandins that have been shown to be synthesised in vivo following COX-facilitated oxygenation of arachidonoyl ethanolamine (anandamide). Although the actions of prostaglandins have been extensively studied, little is known about the physiological or pathophysiological effects of prostamides. Since prostaglandin E₂ has potent immunosuppressive/immunomodulating actions, the aim of the present study was to determine whether the derivative, prostaglandin E₂ ethanolamide (PGE₂-EA), could modulate the production of the pro-inflammatory cytokine tumour necrosis factor-α in human blood and human monocytic cells and indicate whether this action involved the same receptor systems/signals as PGE₂.

Experimental approach

Whole human blood, monocytes isolated from the blood or the human monocytic cell line THP-1 was incubated with LPS and the level of TNF-α produced was measured by ELISA assay. The actions of PGE₂-EA were assessed on the LPS-induced TNF-α release. In addition, in order to ascertain the receptors involved, the levels of cyclic AMP in cells were measured in monocytes and THP-1 cells in response to PGE₂-EA and directly compared to those of PGE₂. The effect of PGE₂-EA on the binding of radiolabelled PGE₂ to cells was also measured. Cells were incubated with radiolabelled arachidonic acid and ethanolamine to estimate the production of PGE₂-EA.

Key results

PGE₂-EA potently suppressed TNF-α production in blood, monocytes and the cell line THP-1 in a concentration-dependent manner. This occurred via cyclic AMP pathways as indicated by agents which interfere with these pathways and also direct ligand binding experiments. It was also shown that the cells were able to endogenously produce PGE₂-EA.

Conclusions and implications

This study reports that PGE₂-EA can downregulate the production of TNF-α by human mononuclear cells in response to an immune stimulus, i.e. LPS-activated TLR4, and that this appears to occur via a cAMP-dependent mechanism that most likely involves binding to the EP₂ receptor.     

Effect of quercetin on Vibrio cholerae induced nuclear factor-κB activation and interleukin-8 expression in intestinal epithelial cells

Vibrio cholerae, the etiological agent of cholera, colonizes the small intestine, produces an enterotoxin and causes acute inflammatory response at intestinal epithelial cell surface. Pretreatment of intestinal epithelial cells with quercetin reduces the level of V. cholerae induced IL-8 in dose and time dependent manner as determined by ELISA and RT-PCR. Immunofluorescence studies showed that quercetin suppresses the translocation of p50 subunit of NF-κB. In vivo, quercetin administration produced a significant reduction of neutrophil infiltration in the intestinal epithelial layer of suckling mouse. Taken together, quercetin could inhibit the V. cholerae induced inflammation and may therefore find use in management of V. cholerae induced pathogenesis.     

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation

Background aimsMesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α–pretreated human bone marrow–derived MSCs on resting or activated T cells.MethodsMSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed.ResultsUnprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions.ConclusionsUnprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo.     

High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-α in lung cancer

INTRODUCTION: Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. RESULTS: Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. CONCLUSION: These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.     

RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1–10 ng/ml) of TNF-α and decreased in high concentration (50–100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.     

G-Protein-coupled receptor agonists differentially regulate basal or tumor necrosis factor-α-stimulated activation of interleukin-6 and RANTES in human airway smooth muscle cells

Summary Using thapsigargin (Tg), an agent that mobilizes calcium by directly emptying intracellular stores, we previously showed that intracellular calcium may play an important role in the regulation of intercellular adhesion molecule (ICAM)-1 gene expression induced by cytokines in human airway smooth muscle (ASM) cells. In the present study, we extended this previous observation by comparing the effect of Tg and other calcium-mobilizing G-protein-coupled receptor (GPCR) agonists on the expression of different pro-inflammatory genes in response to tumor necrosis factor (TNF)-α in ASM cells. We found that in resting cells, Tg (10–100 nM) or the bradykinin (BK) (1–10 μM) and thrombin (Thr) (1 U/ml) stimulated interleukin (IL)-6 secretion but had no effect on regulated on activation, normal T cells expressed and secreted (RANTES) levels. More importantly, such calcium-mobilizing agents significantly enhanced TNF-α-induced IL-6 secretion while RANTES secretion was abrogated. The use of luciferase-tagged IL-6 and RANTES promoter constructs demonstrated similar effects of Tg on IL-6 and RANTES genes in basal and TNF-α-stimulated conditions. The cyclic adenosine monophosphate (cAMP)-dependent pathway plays a minor role in this differential regulation of IL-6 and RANTES genes expression. 2-Aminoethoxydiphenyl borate (APB), a blocker of store-operated calcium channels (SOCs), and bisindolylmaleimide I (Bis I), a broad-spectrum protein kinase C (PKC) inhibitor, inhibited the basal and synergic effects of IL-6 secretion in response to calcium-mobilizing agents and TNF-α, but did not prevent the abrogated effect of RANTES secretion. We also found that Go-6976, a selective calcium-dependent PKC isozyme inhibitor, did not inhibit IL-6 secretion in response to GPCR agonist and TNF-α; whereas Rottlerlin, a PKC-δ inhibitor, inhibited both Thr- and TNF-α-induced expression of IL-6, while BK-induced IL-6 secretion was not affected. Interestingly, TNF-α-induced interferon regulatory factor (IRF)-1 activation was significantly inhibited by all calcium-mobilizing agents, BK, Thr and Tg. These results show that calcium-mobilizing GPCR agonists functionally interact with TNF-α to differentially regulate pro-inflammatory genes expression in human ASM cells, possibly by involving Tg-sensitive intracellular calcium stores, SOC and PKC.     

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.     

Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCγ signaling pathway

Basic fibroblast growth factor (basic FGF) has pivotal roles in the function of various cell types. Here, we report the effects of basic FGF in the regulation of dental pulp stem cell (DPSC) behaviors including maintaining stemness and directing differentiation. Cells isolated from human dental pulp tissues exhibited stem cell properties including the expression of mRNA markers for embryonic and mesenchymal stem cells, the expression of Stro-1, and the multipotential differentiation. Basic FGF stimulated colony-forming units of DPSCs and up-regulated the expression of the embryonic stem cell markers; Oct4, Rex-1, and Nanog. Moreover, osteogenic medium containing basic FGF inhibited alkaline phosphatase enzymatic activity and mineralization of DPSCs. On the contrary, basic FGF appeared to be an influential growth factor in the neurogenic differentiation of DPSCs. In the presence of basic FGF, increased DPSCs neurosphere size and the up-regulation of neurogenic markers were noted. Inhibitors of FGFR or PLCγ were able to ablate the basic FGF-induced neuronal differentiation of DPSCs. Taken together, these results suggest basic FGF may be involved in the mechanisms controlling DPSCs cell fate decisions.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 μmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 μmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.