Release Of Tumor Necrosis Factor Research Articles

Anti-neuroinflammatory Activity of Nobiletin on Suppression of Microglial Activation

A growing body of evidence suggests that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) from the peel of citrus fruits, enhances the damaged cognitive function in disease animal models. However, the neuroprotective mechanism has not been clearly elucidated. Since nobiletin has shown anti-inflammatory effects in several tissues, we investigated whether nobiletin suppresses excessive microglial activation implicated in neurotoxicity in lipopolysaccharide (LPS)-stimulated BV-2 microglia cell culture models. Release of nitric oxide (NO), the major inflammatory mediator in microglia, was markedly suppressed in a dose-dependent manner following nobiletin treatment (1-50 µM) in LPS-stimulated BV-2 microglia cells. The inhibitory effect of nobiletin was similar to that of minocycline, a well-known microglial inactivator. Nobiletin significantly inhibited the release of the pro-inflammatory cytokine tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). LPS-induced phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were also significantly inhibited by nobiletin treatment. In addition, nobiletin markedly inhibited the LPS-induced pro-inflammatory transcription factor nuclear factor κB (NF-κB) signaling pathway by suppressing nuclear NF-κB translocation from the cytoplasm and subsequent expression of NF-κB in the nucleus. Taken together, these results may contribute to further exploration of the therapeutic potential and molecular mechanism of nobiletin in relation to neuroinflammation and neurodegenerative diseases.

Curcumin Potentiates the Antitumor Effects of Bacillus Calmette-Guerin against Bladder Cancer through the Downregulation of NF-κB and Upregulation of TRAIL Receptors

Although Bacillus Calmette-Guerin (BCG) intravesical therapy is a standard treatment for bladder cancer, eventual failure of response is a major problem. Treatments that can augment BCG therapy are urgently needed. We investigated whether curcumin, a component of Curcuma longa (also called turmeric), has potential to improve the current therapy using in vitro and in vivo MBT-2 murine tumor models. We found that curcumin potentiated BCG-induced apoptosis of human bladder cancer cells. BCG stimulated the release of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from peripheral mononuclear neutrophils in a dose- and time-dependent manner, whereas curcumin enhanced the upregulation of TRAIL receptors. Electrophoretic mobility shift assay revealed that curcumin also suppressed the BCG-induced activation of the cell survival transcription factor NF-kappaB. In a syngeneic bladder cancer model, curcumin alone reduced the bladder tumor volume, but a significantly greater reduction was observed when BCG and curcumin were used in combination (P < 0.0001 versus control; P < 0.003 versus BCG alone). This was accompanied by a significant decrease in the proliferation marker Ki-67 (P < 0.01 versus control; P < 0.01 versus BCG alone) and microvessel density (CD31; P < 0.01 versus control; P < 0.01 versus BCG alone), decreased NF-kappaB in tumor tissue compared with the control, induced apoptosis, and decreased cyclin D1, vascular endothelial growth factor, cyclooxygenase-2, c-myc, and Bcl-2 expression in the tumor tissue. Upregulation of TRAIL receptor by the combination was also observed in tumor tissues. Overall, our results suggest that curcumin potentiates the antitumor effect of BCG through the inhibition of NF-kappaB and induction of TRAIL receptors in bladder cancer cells.

Human Natural Killer Cells Are Able to Kill Aspergillus Fumigatus but Not Via the Perforin - Granzyme Pathway.

Abstract 1640Poster Board I-666 IntroductionNatural killer (NK) cells are CD3- CD56+ lymphocytes demonstrating confirmed cytotoxicity against neoplastic and virus infected host cells. Increasing data provide evidence of a direct NK cell effect against extracellular pathogens, such as bacteria, parasites and yeasts, but there is a relative lack of data on their interaction with filamentous fungus and especially with Aspergillus fumigatus. Aspergillus is an omnipresent mold, living in close vicinity with humans, being constantly inhaled in the lungs and thereafter cleared by the innate immune system. Otherwise harmless for healthy people, it is at the origin of invasive Aspergillosis (IA), an extremely devastating disease for immunocompromised subjects. Host's innate immune system controls Aspergillus growth through a complex system of potent effector cells, mediating their antifungal activity mainly by phagocytosis. Our study aims to shed light for the first time on the direct interaction between human NK cells, mediators of extracellular cytotoxicity, and Aspergillus. MethodsNK cells were isolated after magnetic depletion of the peripheral blood of healthy volunteers and they were used after 24h priming with 500 U/ml recombinant interleukin – 2 rhIL-2. To determine gene expression and cytokine release of interferon gamma (IFNg) and Tumor Necrosis Factor- a (TNF-a), NK cells were stimulated for 0, 3, 6 and 12h with different morphologies of Aspergillus: conidia and germlings. To evaluate the lethal impact of NK cells on Aspergillus, plate killing assays were performed at 0, 3 and 6h time points. To illustrate the role of antibody dependent cellular cytotoxicity, ADCC a monoclonal IgG antibody, against germlings, was tested. Transwell permeable membranes, with pores of 0,4 μm, prohibiting the direct contact of cells placed on their opposite sides, but allowing the free circulation of molecules, were used to estimate the effect of cell-fungal contact. To investigate the cytotoxic mechanism involved, NK cells were depleted from perforin and granzymes by treatment with strontium chloride and they had their death ligands, TNF- related apoptosis- inducing ligand (TRAIL) and FasL, neutralised by means of blocking antibodies. The release of cytotoxic granules was estimated by the NK cell surface expression of the marker of degranulation CD107a/b. ResultsObserving the in vitro interaction of NK cells with Aspergillus, fungal germinated morphologies (germlings) showed to be highly immunogenic towards NK cells, compared to conidia, inducing the gene expression and cytokine release of Th1 immune mediators such as IFN-g (p <0,05) and TNF-a.(p <0,1). NK cells demonstrated also a strong lethal impact against germlings (p <0,05). Moreover, the presence of antifungal antibody further potentiated both immunoregulatory and cytotoxic activities. Investigating the means engaged by NK cells to perceive and kill Aspergillus, direct effector–pathogen cell to cell contact was revealed as prerequisite; when this condition was not present there was neither cytokine induction, nor fungal damage (p <0,05). This finding was confirmed by the lack of surface expression of CD107a/b, after NK cell- Aspergillus co-incubation. Investigating the killing pathway we compared the effectiveness of perforin – granzymes depleted NK cells to this of intact cells against germlings and it was found equivalent (p =NS). In a similar way, neutralisation of TRAIL and FasL ligands did not alter the cytotoxic ability of NK cells towards Aspergillus. ConclusionOur data show that human NK cells are stimulated in vitro by Aspergillus germlings, which triggers an immunoregulatory Th1 orientated response and causes important fungal killing. NK cells are not aware of conidia, they are not stimulated by them and par consequence they do not kill them. Finally, we showed that NK cells do not mediate their cytotoxic effect via perforin – granzymes pathway, neither through the engagement of TRAIL, FasL death receptors, suggesting that another pathway is involved in NK cell – Aspergillus fumigatus interplay. We suggest that further investigation of these striking findings might offer a potent immunotherapeutic tool against IA. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia but not cigarette smoke modulates VEGF secretion from human T cells

Vascular endothelial growth factor (VEGF) is an important mitogen with multiple functions. In the present study we investigated whether T cell secreted VEGF and inflammatory cytokines were modulated by cigarette smoke and by a hypoxic microenvironment.T cells from peripheral blood of healthy donors were activated under normoxia (21% O2) or hypoxia (1–2% O2) with or without exposure to cigarette smoke extract. T cells were also obtained from patients with chronic obstructive pulmonary disease (COPD), a smoking-related disease characterized by accumulation of both CD4+ and CD8+T cells.Hypoxia stimulated VEGF secretion from activated T cells, whereas the release of IL-4, IL-6, IL-10, IL-13, IFN-γ and tumour necrosis factor were not altered. Cigarette smoke extract did not affect VEGF secretion neither in hypoxia nor in normoxia, whereas the secretion of all cytokines was inhibited by the extract in both conditions. When recombinant VEGF was added the smoke-induced inhibition of the IFN-γ and IL-13 was not observed. Activated T cells from COPD-patients secreted significantly (p < 0.05) more VEGF compared to T cells from healthy individuals.Our data suggest that both cigarette smoke extract and hypoxia modulate the T cell response. This may be of importance in diseases characterized by T cell accumulation, such as COPD.

Down-regulation of c-FLIP Enhances Death of Cancer Cells by Smac Mimetic Compound

Smac mimetic compounds (SMC) are novel small molecules being developed for cancer therapy. The mechanism of SMC-induced sensitivity in cancer cells depends on autocrine release of tumor necrosis factor alpha (TNFalpha); however, potential mechanisms of resistance remain unknown. Here, we investigated the molecular profile and cytotoxic responsiveness of a diverse panel of 51 cancer cell lines to combinations of a dimeric SMC (AEG40730), death ligand TNFalpha, and tumor necrosis factor-related apoptosis-inducing ligand. Synergy was seen in combination with death receptor agonists in some cells, although single-agent activity was limited to a fewsensitive lines. Unexpectedly, the majority of cell lines resistant to combinations of SMC-AEG40730 and death ligands expressed caspase-8, FADD, RIP1, and ligand receptors necessary for apoptosis execution. Furthermore, TNFalpha-mediated ubiquitination of RIP1 was repressed by SMC-AEG40730 treatment, leading to the formation of the proapoptosis complex II. However, in resistant cancer cells, SMC-AEG40730 repressed TNFalpha-mediated c-jun-NH(2)-kinase activation and the levels of caspase-8 inhibitor c-FLIP were persistently elevated, in contrast to SMC-responsive cancer cells. Importantly, the silencing of c-FLIP restored SMC sensitivity in previously resistant cancer cells by allowing ligand-mediated activation of caspase-8 and caspase-3 to proceed. Together, these results provide mechanistic insight into the action of SMCs, demonstrating that the deciphering of the relevant molecular signature in cancer cells leads to the prediction of cancer cell responsiveness to SMC treatment. Furthermore, a majority of resistant cancer cells were sensitized to SMC-AEG40730 and TNFalpha by down-regulating c-FLIP, suggesting novel approaches in the use of SMCs and c-FLIP antagonists in treating cancer.

Fatty acids as metabolic mediators in innate immunity

Increasing data support the hypothesis of a local and systemic crosstalk between adipocytes and monocytes mediated by fatty acids. The aim of this study was to characterize the immunomodulatory effects of a large panel of fatty acids on cytokines and chemokines in monocytic THP-1 cells and primary human monocytes. We tested whether anti-inflammatory fatty acids are able to inhibit the binding of lipopolysaccharide (LPS) to its receptor, toll-like receptor/MD-2 (TLR4/MD-2). Resistin, monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor (TNF) were measured by enzyme-linked immunosorbent assay. Proteins were analysed by Western blot. A designed Flag-tagged TLR4/MD-2 fusion protein (LPS trap) was used to investigate the effect of fatty acids on binding of LPS to its receptor. In 30 patients with type 2 diabetes mellitus (T2D), the correlation of serum triglyceride levels with LPS-induced monocyte activation was analysed. Eleven fatty acids investigated exerted differential effects on the monocytic release of cytokines and chemokines. Eicosapentaenoic acid had potent anti-inflammatory effects on human primary monocytes and THP-1 cells; 100 and 200 microM eicosapentaenoic acid dose-dependently inhibited LPS binding to the LPS trap. LPS-induced release of monocytic MCP-1 and TNF was significantly and positively correlated with serum triglyceride levels in 30 patients with T2D. Monocytic activation is differentially regulated by fatty acids and depends on triglyceride levels in T2D. The main finding of the present study shows that eicosapentaenoic acid inhibits the specific binding of LPS to TLR4/MD-2. Eicosapentaenoic acid represents a new anti-inflammatory LPS-antagonist.

Rupatadine Inhibits Proinflammatory Mediator Secretion from Human Mast Cells Triggered by Different Stimuli

Background: Mast cells are involved in allergy and inflammation by secreting multiple mediators including histamine, cytokines and platelet-activating factor. Certain histamine 1 receptor antagonists have been reported to inhibit histamine secretion, but the effect on cytokine release from human mast cells triggered by allergic and other stimuli is not well known. We investigated the ability of rupatadine, a potent histamine 1 receptor antagonist that also blocks platelet-activating factor actions, to also inhibit mast cell mediator release. Methods: Rupatadine (1–50 μM) was used before stimulation by: (1) interleukin (IL)-1 to induce IL-6 from human leukemic mast cells (HMC-1 cells), (2) substance P for histamine, IL-8 and vascular endothelial growth factor release from LAD2 cells, and (3) IgE/anti-IgE for cytokine release from human cord blood-derived cultured mast cells. Mediators were measured in the supernatant fluid by ELISA or by Milliplex microbead arrays. Results: Rupatadine (10–50 μM) inhibited IL-6 release (80% at 50 μM) from HMC-1 cells, whether added 10 min or 24 h prior to stimulation. Rupatadine (10–50 μM for 10 min) inhibited IL-8 (80%), vascular endothelial growth factor (73%) and histamine (88%) release from LAD2 cells, as well as IL-6, IL-8, IL-10, IL-13 and tumor necrosis factor release from human cord blood-derived cultured mast cells. Conclusion: Rupatadine can inhibit histamine and cytokine secretion from human mast cells in response to allergic, immune and neuropeptide triggers. These actions endow rupatadine with unique properties in treating allergic inflammation, especially perennial rhinitis and idiopathic urticaria.

Hypoxia but not cigarette smoke modulates VEGF secretion from human T-cells

Vascular endothelial growth factor (VEGF) is an important mitogen with multiple functions. In the present study we investigated whether T cell secreted VEGF and inflammatory cytokines were modulated by cigarette smoke and by a hypoxic microenvironment.T cells from peripheral blood of healthy donors were activated under normoxia (21% O2) or hypoxia (1–2% O2) with or without exposure to cigarette smoke extract. T cells were also obtained from patients with chronic obstructive pulmonary disease (COPD), a smoking-related disease characterized by accumulation of both CD4+ and CD8+T cells.Hypoxia stimulated VEGF secretion from activated T cells, whereas the release of IL-4, IL-6, IL-10, IL-13, IFN-γ and tumour necrosis factor were not altered. Cigarette smoke extract did not affect VEGF secretion neither in hypoxia nor in normoxia, whereas the secretion of all cytokines was inhibited by the extract in both conditions. When recombinant VEGF was added the smoke-induced inhibition of the IFN-γ and IL-13 was not ob...

Impact of Platelets and Fresh Frozen Plasma in Contrast to Red Cell Concentrate on Unstimulated and Stimulated Cytokine Release in an In Vitro Model of Transfusion

Transfusion of blood may contribute to immunomodulation. Leuco-depleted standard blood products are supposed to result in less immunomodulation compared with whole blood. To determine the influence of leuco-depleted blood products on the cytokine response, red blood cell concentrates (RBC), fresh frozen plasma (FFP) and platelet concentrates (PC) were investigated in an in vitro model of blood transfusion. Leuco-depleted standard blood bank RBC, FFP and PC were mixed in vitro with AB0 compatible venous blood from healthy volunteers in ratios of 3:1, 1:1 and 1:3. Specimens were incubated in presence or absence of lipopolysaccharide, 1 mug/ml. After 24 h of incubation cytokine release of tumour necrosis factor (TNF)-alpha and interleukin-10 (IL-10) was measured in cell culture supernatants by means of enzyme-linked immunsorbent assay. Addition of RBC, FFP and PC to venous blood from healthy volunteers led to a significant and dose-dependent increase in spontaneous TNF-alpha and IL-10 release. After endotoxin stimulation, RBC, FFP and PC significantly suppressed the TNF-alpha response, while the stimulated release of IL-10 tended to increase, reaching significance only after high doses of FFP. Addition of leuco-depleted blood products changed the spontaneous and stimulated cytokine response in an in vitro model of transfusion. These data may suggest a possible contribution of transfused FFP and PC to immunomodulation after transfusion similar to RBC.

A TNF- and c-Cbl-dependent FLIPS-degradation pathway and its function in Mycobacterium tuberculosis–induced macrophage apoptosis

Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIP(S), which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIP(S), which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis-triggered macrophage cell death.

Inhibitory effect of saponin fraction from Codonopsis lanceolata on immune cell-mediated inflammatory responses

Saponin components are known to be pharmaceutically, cosmetically and nutraceutically valuable principles found in various herbal medicine. In this study, we evaluated the inhibitory role of saponin fraction (SF), prepared from C. lanceolata, an ethnopharmacologically famous plant, on various inflammatory responses managed by monocytes, macrophages, lymphocytes and mast cells. SF clearly suppressed the release of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha, but not prostaglandin E(2) (PGE(2)). While this fraction did not scavenge the reactivity of SNP-induced radicals in RAW264. 7 cells, it negatively modulated the phagocytic uptake of macrophages treated with FITC-dextran. Interestingly, SF completely diminished cell-cell adhesion events induced by both CD29 and CD43, but not cell-fibronectin adhesion. Concanavalin (Con) A [as well phytohemaglutinin A (PHA)]-induced proliferation of splenic lymphocytes as well as interferon (IFN)-gamma production were also clearly suppressed by SF treatment. Finally, SF also significantly blocked the degranulation process of mast cell line RBL-2H3 cell as assessed by DNP-BSA-induced beta-hexosaminidase activity. The anti-inflammatory activities of SF on NO production seemed to be due to inhibition of nuclear factor (NF)-kappaB activation signaling, since it blocked the phosphorylation of inhibitor of kappaB (IkappaB)alpha as well as inducible NO synthase (iNOS) expression. Therefore, these results suggest that SF may be considered as a promising herbal medicine with potent anti-inflammatory actions.

Ebselen Improves Ischemia-Reperfusion Injury After Rat Lung Transplantation

The heterocyclic organic compound ebselen (2-phenyl-1,2-benizsoselenazol-3(2H)-one) is a glutathione peroxidase mimick with protective properties against oxidative injury. Ebselen also has anti-inflammatory activity, including attenuation of tumor necrosis factor release and increase of interleukin-10, as shown in vivo, in inflammatory and ischemia-reperfusion injuries, including those of the lung. This study was designed to assess its effect on severe ischemia-reperfusion injury in a model of left-sided rat lung isotransplantation. Orthotopic single left-sided lung allotransplantation (Wistar to Wistar) was performed in female rats after a total ischemic time of 18 h. In nine recipients given 500 mg/kg oral ebselen 1 h before transplantation, graft PaO(2)/FiO(2) was improved 24 h after transplantation, as evidenced with a mean (standard deviation) PaO(2) of 139 (61) mmHg vs. eight controls with 65 (33) mmHg (p = 0.009). Bronchoalveolar PMN count was reduced to approximately 50% in the ebselen group compared with controls, whereas no difference in the tumor necrosis factor content was found. We conclude that the improvement of lung function in ebselen-treated transplanted rats is mainly the result of the anti-inflammatory activity of the drug during reperfusion.

Hepatocellular transport and gastrointestinal absorption of lanthanum in chronic renal failure

Lanthanum carbonate is a new phosphate binder that is poorly absorbed from the gastrointestinal tract and eliminated largely by the liver. After oral treatment, we and others had noticed 2-3 fold higher lanthanum levels in the livers of rats with chronic renal failure compared to rats with normal renal function. Here we studied the kinetics and tissue distribution, absorption, and subcellular localization of lanthanum in the liver using transmission electron microscopy, electron energy loss spectrometry, and X-ray fluorescence. We found that in the liver lanthanum was located in lysosomes and in the biliary canal but not in any other cellular organelles. This suggests that lanthanum is transported and eliminated by the liver via a transcellular, endosomal-lysosomal-biliary canicular transport route. Feeding rats with chronic renal failure orally with lanthanum resulted in a doubling of the liver levels compared to rats with normal renal function, but the serum levels were similar in both animal groups. These levels plateaued after 6 weeks at a concentration below 3 microg/g in both groups. When lanthanum was administered intravenously, thereby bypassing the gastrointestinal tract-portal vein pathway, no difference in liver levels was found between rats with and without renal failure. This suggests that there is an increased gastrointestinal permeability or absorption of oral lanthanum in uremia. Lanthanum levels in the brain and heart fluctuated near its detection limit with long-term treatment (20 weeks) having no effect on organ weight, liver enzyme activities, or liver histology. We suggest that the kinetics of lanthanum in the liver are consistent with a transcellular transport pathway, with higher levels in the liver of uremic rats due to higher intestinal absorption.

Aprotinin Modifies Left Ventricular Contractility and Cytokine Release After Ischemia-Reperfusion in a Dose-Dependent Manner in a Murine Model

Periods of ischemia-reperfusion (I/R) during cardiac surgery are associated with transient left ventricular (LV) dysfunction and an inflammatory response. In this study, we examined the potential dose-dependent effects of aprotinin (APRO) on LV contractility and cytokine release in the setting of I/R. An index of LV contractility, LV maximal elastance (E(max)), was measured at baseline, 30 min of ischemia, and 60 min of reperfusion by microtransducer volumetry. Mice were randomized as follows: (a) APRO 20,000 kallikrein-inhibiting units (KIU)/kg (n = 11); (b) APRO 4 x 10(4) KIU/kg (n = 10); (c) APRO 8 x 10(4) KIU/kg (n = 10); and (d) vehicle (saline; n = 10). APRO doses were calculated to reflect half, full, and twice the clinical Hammersmith dosing schedule. After I/R, plasma was collected for cytokine measurements. After I/R, E(max) decreased from the baseline value by more than 40% in the vehicle group as well as in the APRO 4 x 10(4) KIU/kg and APRO 8 x 10(4) KIU/kg groups (P < 0.05). However, E(max) returned to near baseline values in the APRO 2 x 10(4) KIU/kg group. Tumor necrosis factor (TNF) increased 10-fold after I/R, but it was reduced with higher APRO doses. This study demonstrated that a low dose of APRO provided protective effects on LV contractility, whereas higher doses suppressed TNF release. These unique findings suggest that there are distinct and independent mechanisms of action of APRO in the context of I/R.

Glucocorticoid-Induced Tumor Necrosis Factor Receptor–Related Protein Ligand Subverts Immunosurveillance of Acute Myeloid Leukemia in Humans

The reciprocal interaction of tumor cells with the immune system is influenced by various members of the tumor necrosis factor (TNF)/TNF receptor (TNFR) family, and recently, glucocorticoid-induced TNFR-related protein (GITR) was shown to stimulate antitumor immunity in mice. However, GITR may mediate different effects in mice and men and impairs the reactivity of human natural killer (NK) cells. Here, we studied the role of GITR and its ligand (GITRL) in human acute myeloid leukemia (AML). Surface expression of GITRL was observed on AML cells in six of seven investigated cell lines, and 34 of 60 investigated AML patients whereas healthy CD34(+) cells did not express GITRL. Furthermore, soluble GITRL (sGITRL) was detectable in AML patient sera in 18 of 55 investigated cases. While the presence of GITRL was not restricted to a specific AML subtype, surface expression was significantly associated with monocytic differentiation. Signaling via GITRL into patient AML cells induced the release of TNF and interleukin-10 (IL-10), and this was blocked by the inhibition of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2. Furthermore, triggering GITR by surface-expressed and sGITRL impaired NK cell cytotoxicity and IFN-gamma production in cocultures with leukemia cells, and NK cell reactivity could be restored by blocking GITR and neutralization of sGITRL and IL-10. Thus, whereas a stimulatory role of the GITR-GITRL system in mouse antitumor immunity has been reported, our data show that in humans GITRL expression subverts NK cell immunosurveillance of AML. Our results provide useful information for therapeutic approaches in AML, which, like haploidentical stem cell transplantation, rely on a sufficient NK cell response.

1,5-Anhydro- d-fructose attenuates lipopolysaccharide-induced cytokine release via suppression of NF-κB p65 phosphorylation

Lipopolysaccharide (LPS) stimulates macrophages by activating NF-κB, which contributes to the release of tumor necrosis factor (TNF)-α and interleukin (IL)-6. 1,5-anhydro- d-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits anti-oxidant activity and enhances insulin secretion. This study examined the effects of 1,5-AF on LPS-induced inflammatory reactions and elucidated its molecular mechanisms. Before LPS challenge, mice were pretreated with 1,5-AF (38.5 mg/kg). We found that 1,5-AF pretreatment attenuated cytokine release into the serum, including TNF-α, IL-6 and macrophage chemoattractant protein (MCP)-1. Furthermore, pretreatment with 1,5-AF (500 μg/ml) attenuated cytokine release, and 1,5-AF directly inhibited the nuclear translocalization of the NF-κB p65 subunit in LPS-stimulated murine macrophage-like RAW264.7 cells. This inhibition was responsible for decreased LPS-induced phosphorylation on Ser536 of the NF-κB p65 subunit, which is a posttranslational modification involved in the non-canonical pathway. Collectively, these findings indicate that the anti-inflammatory activity of 1,5-AF occurs via inactivation of NF-κB.

RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins

RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

Ca2+-dependent Functions in Peptidoglycan-stimulated Mouse Dendritic Cells

Peptidoglycans (PGN) from bacterial cell walls may modify the course of an infection with bacterial pathogens. The present study explored the effect of PGN on cytosolic Ca²⁺ activity, cytokine production and phagocytosis of mouse dendritic cells (DCs), essential cells in the initiation and direction of antigen-specific T cell responses. Exposure of DCs to PGN was followed by a rapid increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i), which was due to Ca²⁺ release from intracellular stores and influx of extracellular Ca²⁺ across the cell membrane. In DCs isolated from Toll-like receptor 2 (TLR2) deficient mice the effect of PGN on [Ca²⁺]_i was dramatically impaired. The PGN-induced increase of [Ca²⁺]_i was dependent on voltage-gated K⁺ (Kv) channel activity. PGN-induced increase of [Ca²⁺]_i was significantly blunted by margatoxin (MgTx) and perhexiline maleate (PM), inhibitors of Kv1.3 and Kv1.5, respectively. PGN further stimulated the release of tumour necrosis factor α (TNFα), interleukin-12 (IL-12) and interleukin-10 (IL-10), an effect significantly blunted by PM and the specific blocker of store-operated Ca²⁺ channels SKF-96365. Moreover, phagocytic capacity was dramatically increased in PGN-stimulated DCs in the presence of either Kv channel inhibitors or SKF-96365. The observations disclose Ca²⁺ and Kv channel-dependent cytokine production and phagocytosis in PGN-stimulated DCs.

Tumor necrosis factor alpha-dependent aggrecan cleavage and release of glycosaminoglycans in the meniscus is mediated by nitrous oxide-independent aggrecanase activity in vitro

IntroductionLittle is known about factors that induce meniscus damage. Since joint inflammation appears to be a causative factor for meniscal destruction, we investigated the influence of tumor necrosis factor (TNFα) on glycosaminoglycan (GAG) release and aggrecan cleavage in an in vitro model.MethodsMeniscal explant disks (3 mm diameter × 1 mm thickness) were isolated from 2-year-old cattle. After 3 days of TNFα-treatment GAG release (DMMB assay), biosynthetic activity (sulfate incorporation), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR, zymography), and immunostaining of the aggrecan fragment NITEGE were determined.ResultsTNFα induced release of GAG as well as production of NO in a dose-dependent manner, while sulfate incorporation was decreased. TNFα increased matrix metalloproteinase (MMP)-3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 mRNA expression, whereas collagen type I was decreased, and aggrecan, collagen type II as well as MMP-1, -2, -13 and ADAMTS-5 were variably affected. Zymography also showed a TNFα-dependent increase in MMP-3 expression, but pre-dominantly in the pro-form. TNFα-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was induced. Tissue inhibitor of metalloproteinases (TIMP)-3, but not TIMP-1 or -2 inhibited TNFα-dependent GAG release and NITEGE production, whereas inhibition of TNFα-dependent NO generation with the NO-synthetase inhibitor L-NMMA failed to inhibit GAG release and NITEGE production.ConclusionsOur study shows that aggrecanase activity (a) is responsible for early TNFα-dependent aggrecan cleavage and GAG release in the meniscus and (b) might be involved in meniscal degeneration. Additionally, the meniscus is a TNFα-dependent source for MMP-3. However, the TNFα-dependent NO production seems not to be involved in release of proteoglycans under the given circumstances.

Bovine Colostrum Modulates Cytokine Production in Human Peripheral Blood Mononuclear Cells Stimulated with Lipopolysaccharide and Phytohemagglutinin

Bovine colostrum has been shown to influence the cytokine production of bovine leukocytes. However, it remains unknown whether processed bovine colostrum, a supplement popular among athletes to enhance immune function, is able to modulate cytokine secretion of human lymphocytes and monocytes. The aim of this investigation was to determine the influence of a commercially available bovine colostrum protein concentrate (CPC) to stimulate cytokine production by human peripheral blood mononuclear cells (PBMCs). Blood was sampled from four healthy male endurance athletes who had abstained from exercise for 48 h. PBMCs were separated and cultured with bovine CPC concentrations of 0 (control), 1.25, 2.5, and 5% with and without lipopolysaccharide (LPS) (3 microg/mL) and phytohemagglutinin (PHA) (2.5 microg/mL). Cell supernatants were collected at 6 and 24 h of culture for the determination of tumor necrosis factor (TNF), interferon (IFN)-gamma, interleukin (IL)-10, IL-6, IL-4, and IL-2 concentrations. Bovine CPC significantly stimulated the release of IFN-gamma, IL-10, and IL-2 (p < 0.03). The addition of LPS to PBMCs cocultured with bovine CPC significantly stimulated the release of IL-2 and inhibited the early release of TNF, IL-6, and IL-4 (p < 0.02). Phytohemagglutinin stimulation in combination with bovine CPC significantly increased the secretion of IL-10 and IL-2 at 6 h of culture and inhibited IFN-gamma and TNF (p < 0.05). This data show that a commercial bovine CPC is able to modulate in vitro cytokine production of human PBMCs. Alterations in cytokine secretion may be a potential mechanism for reported benefits associated with supplementation.