Alterations in tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 production by natural killer cell-enriched peripheral blood mononuclear cells in chronic alcoholism: relationship with liver disease and ethanol intake

No previous studies have been reported on human alcoholism in which the pattern of cytokine secretion by natural killer (NK) cells is explored. The goal of the present study was to evaluate the role of NK cells in the production of cytokines in patients with chronic alcoholism, analyzing at the same time the possible relationship between cytokine production and both alcoholic liver disease and ethanol (EtOH) intake. A total of 30 chronic alcoholic patients-11 without liver disease [alcoholics without liver disease (AWLD) group] and 19 diagnosed of alcoholic liver cirrhosis-were included in this study. Twenty-five age- and sex-matched healthy volunteers were analyzed as controls. Production of interferon (IFN)-gamma, tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-6 was performed on NK-enriched peripheral blood mononuclear cells (PBMC) after stimulation with IL-2 and IFN-alpha. In AWLD patients, the production of TNF-alpha was significantly reduced, compared with normal controls, under both IFN-alpha (p < 0.01) and IL-2 (p < 0.05) stimulation. In patients with cirrhosis, TNF-alpha production by PBMC enriched in NK cells varied depending on the EtOH intake status at the moment of evaluation. Accordingly, an increased concentration of this cytokine was detected in the supernatants of cirrhotic patients and active EtOH intake, particularly after IFN-alpha stimulation (p < 0.05); whereas, in patients with at least 1 year of alcohol withdrawal, TNF-alpha levels remained within normal range. The results on the production of IL-6 and IFN-gamma in AWLD and cirrhotic patients showed that only cirrhotic patients with a prolonged EtOH withdrawal period display abnormal production. Accordingly, in this group of patients, a significantly increased release of IL-6 was observed after both IFN-alpha and IL-2 stimulation (p < 0.01 and p < 0.05, respectively). By contrast, a lower IFN-gamma production (p < 0.005) was detected with respect to the control group. Our results point to the existence of an abnormal cytokine secretion by NK cells from chronic alcoholism patients, which depend on both the existence of liver disease and the status of EtOH intake.     

Effects of ginkgolides on interleukin-1, tumor necrosis factor-alpha and nitric oxide production by rat microglia stimulated with lipopolysaccharides in vitro

The effects of ginkgolide A (CAS 15291-75-5, BN52020, GA) and B (CAS 15291-77-7, BN52021, GB) on interleukin (IL)-1, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production in resting and lipopolysaccharide (LPS)-stimulated neonatal rat microglia were studied. Apafant (CAS 105219-56-5), a platelet activating factor (PAF) antagonist of triazolobenzodiazepine type was used as control. The biological activities of IL-1 and TNF-alpha were tested by mouse thymocyte proliferation and L929 cytotoxicity assay, respectively. NO concentration was represented by nitrite and determined by Griess reaction. GA 1 nmol/1-10 mumol/l inhibited IL-1 production, and 100 nmol/l-10 mumol/l decreased TNF-alpha and NO production in dose-dependent manner. GB inhibited IL-1, TNF-alpha and NO production at the concentrations 10 nmol/l-10 mumol/l, 100 nmol/l-10 mumol/l and 10 nmol/l-10 mumol/l, respectively. Apafant inhibited IL-1, but not TNF-alpha and NO production. GB plus apafant (50 mumol/l) showed IL-1 and NO inhibitory effects, but not on TNF-alpha. The manner was different from that of GB or apafant alone. The results suggested that GA and GB inhibited proinflammatory cytokines and NO production from LPS-stimulated rat microglia, however, apafant inhibited IL-1 production only. The effects of GA and GB on proinflammatory cytokines and NO production from rat microglia do not seem to be based on PAF receptor antagonism. In addition, GA and GB are regarded as promising agents for the treatment of some neurodegenerative diseases in the central nervous system.     

Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-alpha in rat tissues

Metallothionein (MT) synthesis induced by the inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF), was studied in vivo. Administration of recombinant human IL-6 or TNF to rats caused the acute phase responses including rapid decreases in plasma zinc (Zn), and increases in plasma copper (Cu) and ceruloplasmin. Hepatic concentration of MT-I, one of MT isoforms, began to increase within 3 h after the injection of IL-6 or TNF. In IL-6-treated rats, MT-I concentration in liver reached a maximum level at 12 h and decreased with a transient rebound, whereas, in TNF-treated rats, a high level of MT-I lasted for about 48 h. MT-II, the other MT isoform, was induced more than MT-I in liver by both cytokines. MT-I was also induced in lung and heart by TNF, but little by IL-6. The data suggest that IL-6 may be responsible for MT synthesis in liver, whereas TNF may be responsible not only in liver but also in lung and heart. Furthermore plasma concentration of MT did not always reflect the enhanced concentration of MT by TNF and IL-6 in liver, suggesting involvement of many factors influencing plasma MT levels. The interrelation between IL-6 and TNF for MT synthesis has also been discussed.     

Reinforced cytotoxicity of lymphokine-activated killer cells toward glioma cells by transfection with the tumor necrosis factor-alpha gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human tumor necrosis factor (TNF)-alpha gene by means of novel liposomes with a positive change on their surface. The cells secreted significant amounts of TNF-alpha into the culture medium and exhibited reinforcement of cytotoxicity toward a human glioma cell line (U251-SP), being three times more cytotoxic than nontransfected LAK cells. The mechanism for the reinforcement of cytotoxicity is considered to involve not only an increase in TNF-alpha secretion from LAK cells but also its expression on their surface. Intratumoral or intrathecal injection of LAK cells transfected with the TNF-alpha gene may be useful for the treatment of patients with malignant gliomas.     

Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma

We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.     

Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor-alpha and dexamethasone in human alveolar epithelial cells

Glutathione (GSH) is an important physiological antioxidant in lung epithelial cells and lung lining fluid. We studied the regulation of GSH synthesis in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the anti-inflammatory agent dexamethasone in human alveolar epithelial cells (A549). TNF-alpha (10 ng/ml) exposure increased GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit (gamma-GCS-HS) mRNA at 24 h. Treatment with TNF-alpha also increased chloramphenicol acetyltransferase (CAT) activity of a gamma-GCS-HS 5'-flanking region reporter construct, transfected into alveolar epithelial cells. Mutation of the putative proximal AP-1-binding site (-269 to -263 base pairs), abolished TNF-alpha-mediated activation of the promoter. Gel shift and supershift analysis showed that TNF-alpha increased AP-1 DNA binding which was predominantly formed by dimers of c-Jun. Dexamethasone (3 microM) produced a significant decrease in the levels of GSH, decreased gamma-GCS activity and gamma-GCS-HS mRNA expression at 24 h. The increase in GSH levels, gamma-GCS-HS mRNA, gamma-GCS-HS promoter activity, and AP-1 DNA binding produced by TNF-alpha were abrogated by co-treating the cells with dexamethasone. Thus these data demonstrate that TNF-alpha and dexamethasone modulate GSH levels and gamma-GCS-HS mRNA expression by their effects on AP-1 (c-Jun homodimer). These data have implications for the oxidant/antioxidant balance in inflammatory lung diseases.     

Suppression of B-cell proliferation to lipopolysaccharide is mediated through induction of the nitric oxide pathway by tumor necrosis factor-alpha in mice with acute graft-versus-host disease

Graft-versus-host disease (GVHD) is associated with impaired B-cell responses. We investigated the mechanism of impaired proliferation of B cells in response to the mitogen lipopolysaccharide (LPS) by analyzing the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), both of which have independently been described as important effector mechanisms in the pathogenesis of acute GVHD. A threefold decrease of mature surface Ig-positive (slg+) B cells was observed in GVHD spleens isolated 2 weeks after transplant. However, proliferation of these cells in response to LPS was suppressed by more than 35-fold. Activated GVHD splenocytes secreted large amounts of TNF-alpha and NO in culture. Neutralization of TNF-alpha with anti-TNF-alpha antibody (Ab) both abrogated NO production and restored LPS-induced proliferation of B cells to levels found in non-GVHD control mice. The specific inhibition of NO synthesis with LG-monomethyl-arginine (NMMA) restored splenocyte responses but did not significantly reduce TNF-alpha levels, showing that TNF-alpha per se did not cause immunosuppression. These data show that, during GVHD, induction of the NO pathway is an important mechanism that mediates B-cell hyporesponsiveness to LPS and that this pathway is induced by TNF-alpha.     

Beta-amyloid peptide induces tumor necrosis factor-alpha and nitric oxide production in murine macrophage cultures

We investigated the effect of beta-amyloid peptide (betaA) on the activation of the murine-derived monocyte/macrophage J774 cell-line. BetaA induced tumor necrotic factor-alpha (TNF alpha) in these cells in a dose-dependent manner. Incubation of cells with betaA slightly increased nitric oxide (NO) production, an effect that was significantly enhanced by the addition of interferon-gamma (IFN gamma). Substitution of betaA4 with TFN alpha and incubation of the cultures with IFN gamma resulted in significant NO production, although this was lower than that obtained in the presence of the peptide. Incubation of cultures with a monoclonal antibody (mAb) against TNF alpha abrogated NO production. Our results suggest that betaA4-induced TNF alpha production is a crucial event in the activation of peripheral macrophages.     

Selective induction of tumor necrosis factor receptor type II gene expression by tumor necrosis factor-alpha in C6 glioma cells

The aim of this study, in rabbit tibia, was an evaluation of the early reactions of the tissues to the insertion of polylactic membranes, used in connection with titanium implants. The specimens were retrieved after 1-4 weeks, and a histological analysis was performed. It was possible to see that, in the early implantation phases, no degradation of the macrostructure of the membrane was present. On the outer portion of the membrane many multinucleated giant cells (MGC) were present and membrane fragments were present inside the cytoplasm of these cells. These cells could explain the inflammatory processes reported, in some reports, with the use of materials made by polylactic and polyglycolic acid. We did not observe detrimental effects in the bone tissue around the membrane, and the membrane appeared to have a mechanical stability for the time necessary for bone regeneration.     

Prostaglandin E1 suppresses tumor necrosis factor-alpha and interleukin-10 production by lipopolysaccharides-stimulated mononuclear cells

Previous studies have shown that the intravenous administration of yohimbine, an alpha 2 antagonist, increases norepinephrine turnover and has related anxiogenic effects in humans. We herein report that yohimbine also increases plasma neuropeptide Y (NPY) in healthy human subjects. This finding is consistent with previous reports in animals, but contrasts with a previously reported study in humans. NPY is a 36 amino acid peptide neurotransmitter located in sympathetic and nonsympathetic nerve fibers, as well as in brain structures such as the locus coeruleus, where it is colocalized with norepinephrine. NPY has been shown to inhibit locus coeruleus neuronal firing, decrease norepinephrine release, and increase postsynaptic noradrenergic signal transduction. When administered centrally, NPY also has anxiolytic properties. This study therefore suggests that yohimbine challenge may be useful in assessing NPY and noradrenergic system interactions in neuropsychiatric disorders such as panic disorder or post traumatic stress disorder in which noradrenergic system dysfunction has been observed.     

Oxygen-dependent regulation of energy metabolism in ascites tumor cells by nitric oxide

To understand the role of nitric oxide (NO) in the regulation of energy metabolism of tumor cells, its effect on the respiration and calcium homeostasis was examined with ascites hepatoma (AH130) cells under different oxygen tensions. NO reversibly inhibited the respiration and depolarized the membrane potential of AH130 cells in an oxygen-dependent manner; the inhibition was more marked at physiologically low oxygen concentrations than at its high tensions. NO reversibly decreased the cellular ATP levels and elevated the cytosolic calcium, particularly under low oxygen concentrations. Since the peritoneal cavity is fairly anaerobic, the results suggested that small amounts of NO generated in this compartment might strongly affect the energy metabolism and calcium homeostasis of tumor cells in vivo.     

Responses of human glioblastoma cells to human natural tumor necrosis factor-alpha: susceptibility, mechanism of resistance and cytokine production studies

Responses and susceptibility of 14 human glioblastoma cell lines to human natural tumor necrosis factor-alpha (TNF) were studied in vitro. Susceptibility of glioblastoma cells to TNF varied in experimental conditions applied. Most of glioblastoma cell lines were resistant to cytotoxic activity of TNF in a MTT assay at concentrations below 16 U/ml for 72 h exposure. However, TNF at higher dose, in prolonged exposure and against low density of target cells was antiproliferative for certain glioblastoma cultures. TNF exposure at 10 U/ml for 48 h suppressed DNA synthesis in 9 of 14 glioblastoma cultures, but increased in 3 cultures. In addition, colony forming assay showed anti-clonogenic activity of TNF in 5 of 6 glioblastoma cell lines tested. In spite of their low susceptibility to TNF, glioblastoma cells well responded to TNF stimulation at low dose (10 U/ml) for a short period in the absence of cell damage. Productions of Interleukin-6 (IL-6), IL-8-like activity, granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2 (PGE2) and manganous superoxide dismutase (Mn-SOD) were enhanced or induced by the low-dose TNF stimulation. Mn-SOD, a protein protective against oxidative cell damage, was well induced in time- and dose-dependent manner, however did not correlate with TNF resistance. Whereas the levels of PGE2 in TNF-susceptible cell lines, H-4 and SF-188, were higher than those of other lines. In conclusion, most of glioblastoma cells are resistant to TNF cytotoxic effects, but highly responsive to TNF stimulation. Its effect on glioblastoma cells appears to modulate cell differentiation rather than to kill the cells.     

The effect of x-ray irradiation on the production of interleukin-6 and tumor necrosis factor-alpha by mononuclear cells of the peripheral blood

This study examines the decision of people (N = 56) living in retirement communities to quit driving, and the role of their physician and family in making this decision. Most of the elderly stopped driving when a threshold was reached after an accumulation of compensatory behaviors. Few stopped because of their doctor's advice, although all felt a physician was in the best position to evaluate driving, and family involvement received limited support.     

A potential role for interleukin-15 in the regulation of human natural killer cell survival

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

Bcl-2 is expressed in human natural killer cells and is regulated by interleukin-2

Bcl-2 is a major anti-apoptotic protein expressed in many normal and malignant cells. Recently, low to absent expression was reported in human natural killer (NK) cells cultured in serum-free media which could be induced with stem cell factor. We investigated the expression of bcl-2 protein of NK cells in normal blood donors and compared the bcl-2 expression in CD56+ NK cells with CD3+ T cells. To determine bcl-2 reactivity, a three-color flow-cytometric technique was used. CD56+ CD3- NK cells had an average bcl-2 expression of 83% compared with CD3+ T cells. CD56 and CD3 double positive T cells had an average content of 111% compared with all peripheral CD3+ T lymphocytes. When peripheral mononuclear cells were cultured with interleukin-2 (IL-2), bcl-2 could be upregulated by IL-2 in all cell populations studied. The induction of bcl-2 in these cell populations paralleled the induction in CD56- T lymphocytes cultured under identical conditions. The induction of bcl-2 by IL-2 was confirmed by Western blotting. The maximum induction of bcl-2 by IL-2 was observed at an IL-2 dose of 100-1,000 U/ml. Our data confirm the anti-apoptotic protein bcl-2 as an activation- or proliferation-associated marker of normal NK cells which can be induced by IL-2.