Oral administration of milk fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 to DBA/1 mice inhibits secretion of proinflammatory cytokines

We have previously shown that oral administration of skimmed milk(SM) fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1/SM) to DBA/1 mice inhibited the development of collagen-induced arthritis (CIA). In this study, our aim was to examine possible mechanisms of inhibiting the development of CIA. We studied the effect of OLL1073R-1/SM on cytokine secretion from cells of popliteal lymph nodes (lymph node cells; LNC) of mice. The results showed that feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines such as interferon γ (IFN-γ), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and the chemokine, monocyte chemoattractant protein 1 (MCP-1). The most prominent effect was inhibition of TNF-α. Secretion of IL-2 and IL-4 were not influenced. Feeding OLL1073R-1/SM inhibited secretion of proinflammatory cytokines produced by accessory cells, but not T cells. We conclude that CIA may be prevented via down regulation of secretion of proinflammatory cytokines such as IL-6, TNF-α and IFN-γ, and of the chemokine of MCP-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Does acetylcholinesterase secretion involve an ADAMs-like metallosecretase?

Summary The ADAMs (A Disintegrin And Metalloprotease-like) family is a large and rapidly expanding group of metallo-proteinases with structural similarity. The are normally characterized by the presence of a proteolytic domain and disintegrin and signalling domains. Although 21 ADAMs proteins have been already cloned to date, in most cases their natural substrates are unknown. The best characterized representative of the mammalian ADAMs family is the TNF-α converting enzyme (TACE). TACE is an integral membrane metalloproteinase that causes the secretion of the active form of TNF-α from its plasma membrane precursor and thus can be regarded as a membrane protein secretase. Secretion of membrane proteins is a very well documented biological phenomenon and was demonstrated for a diverse range of membrane proteins, two examples being angiotensin converting enzyme (ACE) and Alzheimer's amyloid precursor protein (APP). ACE and APP secretion was shown to possess substantial similarity with the secretion of TNF-α. In the present study, we have attempted to demonstrate that a metalloproteinase might be involved in the shedding of another membrane-bound protein—acetylcholinesterase (AChE). Secretion of AChE by human neuroblastoma SH-SY5Y cells was found to be inhibited by a selective hydroxamate metalloproteinase inhibitor batimastat (20 μM), and stimulated by carbachol (20 μM), which have previously been shown to regulate the activity of APP α-secretase in a similar manner. The role of ADAMs proteins in the shedding of molecules from the cell surface is discussed.     

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.     

Interferon-γ- and lipopolysaccharide-induced tumor necrosis factor-α is required for nitric oxide production: Tumor necrosis factor-α and nitric oxide are independently involved in the killing ofMycobacterium microti in interferon-γ-and lipopolysaccharide-treated J774A.1 cells

A comparative study was done using J774A.1 and J774A. 1-derived transfected cells (J774A.1 C.1) containing antisense tumor necrosis factor α (TNF-α) plasmid to determine the role of endogenous TNF-α on nitric oxide production as well as on the growth ofMycobacterium microti in interferon γ (IFN-γ)- and lipopolysaccharide (LPS)-treated cells. On stimulation with IFN-γ and LPS a higher level of NO was observed in J774A.1 cells compared to J774A.1 C.1 which indicated that endogenous TNF-α is required for the production of NO. Comparing the effect of IFN-γ and LPS on the intracellular growth ofM. microti, the growth-reducing activity was higher in J774A.1 cells than in J774A.1 C.1 cells and was not completely abrogated in the presence of the nitric oxide inhibitorN ^G-methyl-l-arginine (l-NMA). J774A.1 C.1 cells infected withM. microti produced a significant amount of NO when exogenous TNF-α was added along with IFN-γ and LPS and the concentration of intracellular bacteria decreased almost to that in IFN-γ and LPS treated parental J774A.1 cells. Addition of exogenous TNF-α even in the presence ofl-NMA in J774.1 C.1 cells could also partially restore intracellular growth inhibition ofM. microti caused by IFN-γ and LPS. TNF-α is probably required for the production of NO in J774A.1 cells by IFN-γ and LPS but TNF-α and NO are independently involved in the killing of intracellularM. microti with IFN-γ and LPS.     

H2O2-induced secretion of tumor necrosis factor-α evokes apoptosis of cardiac myocytes through reactive oxygen species-dependent activation of p38 MAPK

P38 mitogen-activated protein kinases (p38 MAPK) and tumor necrosis factor-α (TNF-α) play important roles in oxidative stress-induced apoptosis in cardiac myocytes. However, the regulation and functional role of cross-talk between p38 MAPK and TNF-α pathways have not yet been fully characterized in cardiac myocytes. In this study, we found that inhibition of p38 MAPK with SB-203580 (SB) reduced H₂O₂-stimulated secretion of TNF-α, whereas pre-activation of p38 MAPK with sodium arsenite (SA) enhanced H₂O₂-stimulated secretion of TNF-α. In addition, pretreatment of cells with TNF-α increased basal and H₂O₂-stimulated p38 MAPK and apoptosis of cardiac myocytes, and p38 MAPK-associated apoptosis of cardiac myocytes induced by TNF-α was blocked by inhibition of p38 MAPK with SB. Finally, H₂O₂-induced apoptosis was attenuated by the inhibitors of p38 MAPK or reactive oxygen species (ROS), whereas it was enhanced by p38 MAPK agonist SA. These results suggest that H₂O₂-induced secretion of TNF-α increases apoptosis of cardiac myocytes through ROS-dependent activation of p38 MAPK. This may represent a novel mechanism that TNF-α partly interplays with p38 MAPK pathways during oxidative stress-modulated apoptosis in cardiac myocytes.     

IFN-α regulates IL 10 production by CML cells in vitro

High levels of spontaneous in vitro IL 10 secretion by a subset of untreated chronic phase CML patients' cells are shown to be decreased in the presence of IFN-α. However, the lower level of spontaneous IL 10 secretion by healthy control cells are was not depressed by IFN-α. In contrast to its effects on IL 10 production, IFN-α increased the low spontaneous secretion of IL 1α by patients' cells, bud did not further increase the higher levels of spontaneous IL 1β secretion by normal cells. It had no effect on secretion of TNF-α by patients or normals. Spontaneous secretion of IL-1α (or IFN-γ) by patients' cells was not observed whether or not IFN-α was present. Therefore, one mechanism of action of IFN-α in vivo may involve decreasing endogenous IL 10 secretion (thereby reducing suppressive effects on T cell reactivity) and increasing IL 1β secretion (thereby enhancing antigen presentation). Received: November 1998 / Accepted: 1 March 1999     

The contact-mediated response of peripheral-blood monocytes to preactivated T cells is suppressed by serum factors in rheumatoid arthritis

Stimulation of monocytes/macrophages after cell contact with preactivated T cells has been suggested to contribute to the excessive TNF-α production in rheumatoid arthritis (RA). In this study, T cell-contact-dependent TNF-α production by peripheral-blood monocytes in vitro was investigated and found to be significantly lower in treated and untreated patients with RA than in healthy controls. This suppression was not due to a general deficiency of monocytes to respond, because responses to lipopolysaccharide were comparable in patients and controls. In agreement with the pivotal role of TNF-α in RA, T cell-dependent induction of TNF-α in synovial macrophages was fivefold to tenfold higher than in peripheral-blood monocytes from either patients or controls. The decreased response of peripheral-blood monocytes from patients with RA was found to be mediated by inhibitory serum factors, because the addition of patient sera to monocytes from healthy controls suppressed TNF-α response in the co-culture assay. Preincubation of monocytes from healthy controls with RA serum was sufficient to suppress the subsequent TNF-α response in T cell co-cultures, indicating that inhibitory factors do indeed bind to monocyte surfaces, which might represent a regulatory counter-action of the immune system to the long-standing and consuming autoimmune process in RA. There are some indications that apolipoprotein A-1 might be part of this regulatory system.     

Pim protein kinase-3 is regulated by TNF-α and promotes endothelial cell sprouting

Tumor necrosis factor-α (TNF-α) plays an important role in pathological angiogenesis associated with inflammatory response. Pim-3 kinase belonging to serine/threonine protein kinases is a potent suppressor of myc-induced apoptosis. We have recently demonstrated that Pim-3 plays an essential role in endothelial cell (EC) spreading and migration. In this study, we showed that TNF-α transiently increased Pim-3 mRNA expression, and this was mediated through Tumor necrosis factor-α receptor-1 (TNFR1) pathway in ECs. TNF-α could promote stabilization of Pim- 3 mRNA in ECs. Small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired TNF-α-induced formation of EC membrane protrusions in vitro. Furthermore, Pim-3 silencing inhibited EC sprouting in subcutaneous Matrigel in vivo. eNOS mRNA abundance was lower in Pim-3 siRNA transfected ECs compared with the control ECs. These observations suggest that Pim-3 plays a role in TNF-α-induced angiogenesis.     

Activation of EGF receptor family members suppresses the cytotoxic effects of tumor necrosis factor-α

Tumor necrosis factor (TNF)-α has a broad range of biological activities, which depend heavily on cell type and physiological condition. In a panel of human tumor cell lines we analyzed expression of the receptor tyrosine kinases EGFR, ErbB2 and ErbB3, and the response to TNF-α. Among the cell lines tested those resistant to TNF-α were found to express high levels of either EGFR, or ErbB2 and ErbB3. In TNF-sensitive breast and cervical carcinoma cells activation of EGFR or ErbB2 by the exogenous growth factors EGF and heregulin β1 resulted in a significant increase in the number of cells surviving TNF-α treatment. In contrast, inhibition of EGFR activation in TNF-resistant breast carcinoma cells by the novel antagonistic anti-EGFR antibody 14E1 sensitized the cells to the cytotoxic effects of TNF-α. A bacterially expressed fusion protein consisting of a 14E1 single-chain (sc) Fv antibody fragment linked to human TNF-α retained TNF-α activity. This scFv(14E1)-TNF-α molecule localized specifically to EGFR on the surface of tumor cells and activated the NF-κB pathway in co-cultured T cells, as demonstrated by electrophoretic mobility shift assays. Received: 6 May 1998 / Accepted: 16 July 1998     

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.     

Gypenoside XLIX isolated from Gynostemma pentaphyllum inhibits nuclear factor-kappaB activation via a PPAR-alpha-dependent pathway

Summary Nuclear factor (NF)-κB is important in the generation of inflammation. Besides regulating lipid metabolism, peroxisome proliferator-activated receptor (PPAR)-α activators also reduce NF-κB activation to terminate activation of inflammatory pathways. Gynostemma pentaphyllum (GP) has been used to treat various inflammatory diseases and hyperlipidemia. Here, we demonstrate that GP extract and one of its main components, Gypenoside XLIX (Gyp-XLIX) inhibited LPS-induced NF-κB activation in murine macrophages. Furthermore, Gyp-XLIX restored the LPS- and TNF-α-induced decrease in cytosolic I-κBα protein expression and inhibited the translocation of NF-κB(p65) to the nucleus in THP-1 monocyte and HUVEC cells. The inhibition of LPS- and TNF-α-induced NF-κB luciferase activity in macrophages was abolished by MK-886, a selective PPAR-α antagonist. GP extract and Gyp-XLIX (EC₅₀: 10.1 μM) enhanced PPAR-α luciferase activity in HEK293 cells transfected with the tK-PPREx3-Luc reporter plasmid and expression vectors for PPAR-α. Additionally, Gyp-XLIX specifically enhanced PPAR-α mRNA and protein expression in THP-1-derived macrophage cells. The selectivity of Gyp-XLIX for PPAR-α was demonstrated by the activation of only PPAR-α in HEK293 cells transfected with expression vectors for PPAR-α, PPAR-β/δ or PPAR-γ1 plasmids and in THP-1-derived macrophage naturally expressing all three PPAR isoforms. The present study demonstrates that Gyp-XLIX, a naturally occurring gynosaponin, inhibits NF-κB activation via a PPAR-α-dependent pathway.Tom Hsun-Wei Huang and Yuhao Li contributed equally.     

A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth

Intravesical bacillus Calmette-Guerin (BCG) therapy is considered the most successful immunotherapy against solid tumors of human bladder carcinoma. To determine the actual effector cells activated by intravesical BCG therapy to inhibit the growth of bladder carcinoma, T24 human bladder tumor cells, expressing very low levels of class I MHC, were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) with live BCG. The proliferation of T24 cells was markedly inhibited when BCG-infected dendritic cells (DCs) were added to the culture although the addition of either BCG or uninfected DCs alone did not result in any inhibition. The inhibitory effect was much stronger when the DCs were infected with live BCG rather than with heat-inactivated BCG. The live BCG-infected DCs secreted TNF-α and IL-12 within a day and this secretion continued for at least a week, while the heat-inactivated BCG-infected DCs secreted no IL-12 and little TNF-α. Such secretion of cytokines may activate innate alert cells, and indeed NKT cells expressing IL-12 receptors apparently proliferated and were activated to produce cytocidal perforin among the PBMCs when live BCG-infected DCs were externally added. Moreover, depletion of γδ T-cells from PBMCs significantly reduced the cytotoxic effect on T24 cells, while depletion of CD8β cells did not affect T24 cell growth. Furthermore, the innate effectors seem to recognize MICA/MICB molecules on T24 via NKG2D receptors. These findings suggest the involvement of innate alert cells activated by the live BCG-infected DCs to inhibit the growth of bladder carcinoma and provide a possible mechanism of intravesical BCG therapy.     

Rubella vaccine-induced cellular immunity: evidence of associations with polymorphisms in the Toll-like, vitamin A and D receptors, and innate immune response genes

Toll-like, vitamin A and D receptors and other innate proteins participate in various immune functions. We determined whether innate gene-sequence variations are associated with rubella vaccine-induced cytokine immune responses. We genotyped 714 healthy children (11–19 years of age) after two doses of rubella-containing vaccine for 148 candidate SNP markers. Rubella virus-induced cytokines were measured by ELISA. Twenty-two significant associations (range of P values 0.002–0.048) were found between SNPs in the vitamin A receptor family (RARA, RARB, TOP2B and RARG), vitamin D receptor and downstream mediator of vitamin D signaling (RXRA) genes and rubella virus-specific (IFN-γ, IL-2, IL-10, TNF-α, and GM-CSF) cytokine immune responses. A TLR3 gene promoter region SNP (rs5743305, −8441A > T) was associated with rubella-specific GM-CSF secretion. Importantly, SNPs in the TRIM5 gene coding regions, rs3740996 (His43Tyr) and rs10838525 (Gln136Arg), were associated with an allele dose-related secretion of rubella virus-specific TNF-α and IL-2/GM-CSF, respectively, and have been previously shown to have functional consequences regarding the antiviral activity and susceptibility to HIV-1 infection. We identified associations between individual SNPs and haplotypes in, or involving, the RIG-I (DDX58) gene and rubella-specific TNF-α secretion. This is the first paper to present evidence that polymorphisms in the TLR, vitamin A, vitamin D receptor, and innate immunity genes can influence adaptive cytokine responses to rubella vaccination.     

Antitumor therapeutic effects of a genetically engineered <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> harboring TNF-α in mice

Although the use of TNF-α in the treatment of cancer is restricted due to its non-specific cytotoxicity and narrow range of applications to different cancers in clinical trials, we investigated a safe anti-cancer drug by the use of engineered bacterial capsule harboring TNF-α. The engineered bacterial capsule was designed to target cancer cells, promote a tumor-suppressive environment, and increase the efficacy of existing cancer treatments, including chemotherapy, radiotherapy, and cell therapy. The engineered bacterial capsule was constructed with Salmonella capsulizing TNF-α protein, which was produced and capsulized by Salmonella to reduce side effects of the protein. This bacterial capsule induced a tumor-suppressive environment through the activation of natural killer cells. Engineered bacterial capsule invaded tumor cells, released TNF-α, and induced apoptosis of tumor cells without apparent side effects. In a murine melanoma model, the bacterial capsule of TNF-α significantly inhibited tumor growth by 80–100% and prolonged the survival of the mice. When tested in combination with chemotherapy (cisplatin), antibiotics, and vaccine, recombinant microbial treatment increased the anti-tumor effects of existing therapies. The anti-tumor effects of the bacterial capsule of TNF-α were also observed in cervical cancer, melanoma, breast cancer, colon cancer, and renal carcinoma. These results suggest that the bacterial capsule of TNF-α is a promising strategy for TNF-α treatment.     

Short term protective effects of iron in a murine model of ischemia/reperfusion

The role of iron in the pathogenesis of cardio-vascular disorders is still controversial. We studied the effects of iron perturbations on myocardial injury upon temporary ischemia/reperfusion. C57BL/6J male mice were injected with iron dextran for 2 weeks while controls received saline. Mice were then subjected to 30 min of myocardial ischemia and subsequent reperfusion for 6–24 h. Tissue damage was quantified histologically and by troponin T determination. The expressions of tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were investigated in non-ischemic and ischemic regions of both groups. After myocardial ischemia and reperfusion, troponin T levels, as a marker of myocardial damage, were significantly reduced in iron-treated mice as compared to control mice (P < 0.05). Under the same conditions the infarction area and damage score were significantly lower in iron-treated animals. In parallel, TNF-α and SOD expressions were increased in infarcted regions of iron-treated mice as compared to controls, whereas myocardial iNOS expression was significantly lower in iron-treated mice. Although, iron challenge increased radical formation and TNF-α expression in vivo, this did not result in myocardial damage which may be linked to the parallel induction of SOD. Importantly, iron treatment inhibited iNOS expression. Since, an increased nitric oxide (NO) formation has been linked to cardiac damage after acute myocardial infarction, iron may exert short time cardio-protective effects after induction of ischemia/reperfusion via decreasing iNOS formation. Both authors contributed equally to this work.