Y-24180, an antagonist of platelet-activating factor, suppresses interleukin 5 production in cultured murine th(2)cells and human peripheral blood mononuclear cells

Interleukin (IL)-5 has been shown to play an essential role in the pathogenesis of airway inflammation. We investigated the effect of 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6, 9-dimethyl-6 H -thieno[3,2- f ][1,2,4]triazolo[4,3- a][1,4]diazepine (Y-24180), an antagonist of platelet-activating factor (PAF), on the production of IL-5 in cultured D10.G4.1 cells, a murine Th(2)clone, and human peripheral blood mononuclear cells (PBMC). As a result, Y-24180 was found to suppress both the mRNA expression of IL-5 and the subsequent secretion of this cytokine in antigen-stimulated D10.G4.1 cells. Y-24180 also suppressed the production of IL-4, another Th(2)type cytokine, at the level of mRNA expression, however, it hardly affected the mRNA expression for IL-6 or IL-10, thus indicating it to have a selective action against IL-5 and IL-4. The suppressive effect of Y-24180 on the secretion of IL-5 by human PBMC was more potent than that of WEB2086, which is another PAF-antagonist. These results suggest that Y-24180 suppresses IL-5 production through a common pathway which also affects the production of IL-4, even though the mechanism remains to be elucidated as to whether the PAF-antagonistic actions are involved or not.     

FK506 suppresses E-selectin, ICAM-1 and VCAM-1 expression on vascular endothelial cells by inhibiting tumor necrosis factor alpha secretion from peripheral blood mononuclear cells

FK506 suppresses activation of T cells; however, it down-regulates E-selectin, ICAM-1 and VCAM-1 expression in inflamed tissues. In this study, we investigated the effect of FK506 on expression of those adhesion molecules on human vascular endothelial cells (HMVEC). Culture supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD2 antibodies effectively induced the expression of E-selectin, ICAM-1 and VCAM-1 on HMVEC, and treatment with FK506 down-regulated their expression. Culture supernatant contained tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta, which effectively induced adhesion molecules, and FK506 suppressed both cytokine secretions. TNFalpha content in culture supernatant was parallel to the induction of adhesion molecules by the culture supernatant. IL-1beta content was not enough to induce those adhesion molecules. Anti-TNFalpha antibody completely inhibited those expressions. FK506 did not inhibit either TNFalpha- or IL-1beta-induced expression of adhesion molecules, or viability of HMVEC. These results indicate that FK506 suppresses migration of inflammatory cells through the inhibition of TNFalpha secretion from leukocytes.     

Enhanced phosphorylation of 65 and 74 kDa proteins by tumor necrosis factor and interleukin-1 in human peripheral blood mononuclear cells

Tumor necrosis factor (TNF) and interleukin-1 (IL-1) enhanced the phosphorylation of identical cytosolic 65 kDa protein (P65 or l-plastin) and 74 kDa protein (P74) at serine residues in human peripheral blood mononuclear cells (PBMC). The isoelectric points of P65 and P74 were 5.6 and 4.7 to 5.0, respectively. The phosphorylation of these proteins increased with a few minutes and reached maximal levels of approximately 3 times the unstimulated levels by 10 minutes. The phosphorylation of P65 and P74 was extensively enhanced by a potent protein kinase C (PKC) activator, PMA. However, there was no translocation of PKC from cytosol to membrane in PBMC that was stimulated with either TNF or IL-1, which suggests that PKC does not participate in TNF or IL-1 signal transduction. cAMP dependent protein (PKA) activators, forskolin and PGE2, failed to increase the phosphorylation, which is in agreement with the data showing that neither TNF nor IL-1 increased cAMP levels in PBMC. These results suggest that induction of phosphorylation of P65 and P74 by TNF and IL-1 is not mediated by PKC and PKA but may be mediated by another protein kinase and result in overlapping of biological activities between TNF and IL-1.     

Transforming growth factor beta induces the production of interleukin 6 by human peripheral blood mononuclear cells

Previous studies have indicated that the cytokine transforming growth factor beta 1 (TGF beta 1) has immunosuppressive properties and can inhibit the production of tumor necrosis factor (TNF) and Interleukin 1 (IL 1) by human peripheral blood mononuclear cells. In this study, we have examined the effects of TGF beta 1 on the production of Interleukin 6 (IL 6) by human peripheral blood mononuclear cells. Treatment with only TGF beta 1 leads to the induction of IL 6, and this was both dose- and time-dependent. The effect of TGF beta 1 was evident at the level of IL 6 mRNA, suggesting TGF beta 1-induced de novo synthesis of IL 6. Induction of IL 6 by TGF beta 1 was specific, as other cytokines made by mononuclear cells (TNF and IL 1) were not induced by TGF beta 1. Furthermore, when a panel of stimuli were compared for their ability to induce IL 1, TNF and IL 6 in the presence or absence of TGF beta 1, IL 6 levels were augmented in the presence of TGF beta 1, while the induction of IL 1 and TNF was inhibited significantly. These results indicate that TGF beta 1 has complex effects on the production of cytokines by peripheral blood mononuclear cells and that TGF beta 1 is not inhibitory for all cytokine production. The ability of TGF beta 1 to induce IL 6 suggests that IL 6 may mediate some of the effects of TGF beta 1.     

Effect of interleukin-2 on synthesis of B cell activating factor belonging to the tumor necrosis factor family (BAFF) in human peripheral blood mononuclear cells

B cell activating factor belonging to the tumor necrosis factor family (BAFF) is a cytokine, indispensable for B cell survival, maturation, and activation. Over-expression of BAFF leads to lupus like disease in mice and the serum level of BAFF is elevated in human lupus. However, little is known about BAFF synthesis and its regulation. In this study, we examined the effects of a series of inflammatory cytokines on BAFF production in human peripheral blood mononuclear cells (PBMCs) in vitro. We found interleukin-2 (IL-2) strongly and dose-dependently stimulated BAFF synthesis in PBMCs, and an anti-IL-2 antibody neutralized the effect. Furthermore, T and NK cells produced BAFF with IL-2 stimulation. From these observations, IL-2 is one of the regulatory cytokines having a positive effect on BAFF synthesis in human peripheral T and NK cells. Persistent over-production of IL-2 might lead to up-regulation of BAFF synthesis in PBMCs in pathological conditions such as lupus.     

Hypoxia increases production of interleukin-1 and tumor necrosis factor by human mononuclear cells

Exposure to hypoxia (PO2 = 9 +/- 1 torr) increased human peripheral blood mononuclear cell production and secretion of interleukin-1 (IL-1)alpha, IL-1 beta, and tumor necrosis factor (TNF) percent of control = 190% for IL-1 alpha, p = 0.014; 219% for IL-1 beta, p = 0.014; and 243% for TNF, p = 0.037) following treatment with endotoxin (1 ng/ml). Hypoxia potentiated the increased production of these inflammatory cytokines at subthreshold levels of endotoxin with potentiation increasing at lower O2 concentrations. Hypoxia also increased cytokine production induced by the tumor promoter phorbol myristate acetate, suggesting a generalized biologic response. We conclude that hypoxia increases IL-1 and TNF production and speculate that this mechanism aggravates a variety of pathologic conditions involving endotoxin such as adult respiratory distress syndrome (ARDS), multiple organ failure, and septic shock.     

Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon

We have used the quantitative binding of murine monoclonal antibodies to the surface of cultured human umbilical vein endothelial (HUVE) cells to study the responses of HUVE cells to three different immune mediators: interleukin 1 (IL 1), tumor necrosis factor (TNF), and immune interferon (IFN-gamma). Antibody H4/18, reactive with an endothelial cell-specific activation antigen, does not bind to unstimulated HUVE cells but shows rapidly and transiently inducible binding (peak 4 to 6 hr) to cells stimulated by IL 1 or TNF that declines to basal levels by 24 hr, even in the continued presence of mediator. Binding of H4/18 is unaffected by IFN-gamma. Antibody RR1/1, reactive with intercellular adhesion molecule 1, binds to unstimulated HUVE cells, but binding is rapidly increased (plateau 24 hr) after stimulation by IL 1 or TNF and slowly increased (over several days) by IFN-gamma. In contrast to H4/18 binding, the increase in RR1/1 binding is sustained in the continued presence of mediator. Antibody W6/32, reactive with HLA-A,B antigens, binds to unstimulated HUVE cells and shows gradually progressive increases (over several days) in binding upon treatment with IFN-gamma or TNF. These observations demonstrate that HUVE cells show distinct but overlapping patterns of antigenic modulation in response to three different lymphokines, and suggest that the "activation" of endothelial cells observed in situ may represent a complex integration of several lymphokine-mediated signals.     

High expression of tumor necrosis factor alpha receptors in peripheral blood mononuclear cells of obese type 2 diabetic women

The tumor necrosis factor system plays an important role in the pathogenesis of obesity and type 2 diabetes (DM), by a complex and only partially understood mechanism. In this study we analyze the mRNA expression levels of TNFalpha and its receptors (TNFR1 and TNFR2), in peripheral blood mononuclear cells (PBMC) from eleven, non-morbid, obese and 14, obese, type 2 DM women, by real-time quantitative PCR. We show an increase in the TNFR2 to TNFR1 ratio (mTNFR2/mTNFR1) in type 2 DM (r = 0.63; p = 0.021, after adjusting for age). Likewise, a positive correlation between mTNFR2/mTNFR1 and glucose was observed (r = 0.5; p = 0.029) in the whole group. We performed an oral glucose tolerance test with 75 g of glucose in obese, non-diabetic women in order to evaluate the effect of an acute glucose increase on the tumor necrosis factor system at 60 min and 120 min. We show that except for a positive association of mTNFR1 with body mass index at 60 min and of mTNFR2 with plasmatic triglycerids levels, no other significant differences were elicited by acute glucose in obese, non-diabetic women. These findings are in agreement with a functional role for the TNF system in obese women in obesity-linked, type 2 diabetes. Copyright John Libbey Eurotext 2003.     

Evidence for an endothelial cell-derived factor which stimulates the growth of human peripheral blood mononuclear cells

Evidence is provided which demonstrates that conditioned media of cultured endothelial cells derived from human umbilical veins contained a factor which stimulated peripheral blood mononuclear cell [3H]thymidine uptake. A dose-dependent response in peripheral blood mononuclear cell [3H]thymidine uptake was obtained when cells were incubated with increasing concentrations of supernatant of endothelial cell cultures. Studies on temporal kinetics demonstrated that stimulatory activity was evident when mononuclear cells had been incubated with endothelial cell supernatant for 120 hr or more. Preliminary characterization showed the growth immunoregulatory factor to have a molecular weight greater than 100,000 Da.     

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.     

Ovine interleukin 12 has biological activity on ovine and human activated peripheral blood mononuclear cells

Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits that form the biologically active p70 molecule, and is a potent inducer of the pro-inflammatory cytokine IFN-gamma. In this study the coding sequence for ovine interleukin 12 p35 and p40 subunits was derived by RT-PCR cloning. Ovine p35 and p40 cDNA sequences show a high level of similarity at the nucleic acid and protein levels when compared to corresponding bovine and human sequences. In particular, cysteine residues and N-linked glycosylation sites are conserved between species. Secretion of the IL-12 heterodimer from CHO cells co-transfected with ovine p35 and p40 cDNA was shown by immunoprecipitation of a 60 and 66 kDa protein from transfectant supernatant. In addition, the supernatant from co-transfected cells augmented the proliferation of Con A-activated ovine peripheral blood mononuclear cells (PBMNC). Cross-species activity was shown by the enhancement of proliferation of human phytohaemagglutinin (PHA)-activated PBMNC. Supernatants from co-transfectants of hu p35/ov p40 and ov p35/hu p40, to generate chimeric heterodimers, also demonstrated stimulatory activity. Human and chimeric IL-12-induced proliferation of activated PBMNC was inhibited using an anti-human IL-12 polyclonal antibody, however this antibody showed minimal inhibition of ovine IL-12. This study suggests that ovine IL-12 has biological properties similar to its human counterpart.     

Conjugated linoleic acid can prevent tumor necrosis factor gene expression by inhibiting nuclear factor binding activity in peripheral blood mononuclear cells from weaned pigs challenged with lipopolysaccharide

Twenty-four weanling barrows were fed corn-soybean diets supplemented with 2% conjugated linolenic acid (CLA) or soybean oil. On day 14 and 21, pigs were injected intraperitoneally with lipopolysaccharide (LPS) or sterile saline. Plasma samples were collected 2h after injection. Peripheral blood mononuclear cells (PBMC) were also collected on day 21, 2 h after injection to determine tumor necrosis factor-alpha (TNF-alpha) production and its mRNA expression. The results indicate that dietary CLA inhibited the production of TNF-alpha by pig PBMC both at the protein and mRNA expression level. In a second experiment, PBMC, collected from a healthy pig, were incubated with either c9,t11-CLA or t10,c12-CLA, or without CLA and stimulated with LPS. Both CLA isomers inhibited LPS-stimulated TNF-alpha production and expression, which may be partially due to inhibition of the binding activity of nuclear factor-kappaB. The t10,c12 isomer was more effective than the c9,t11-CLA isomer in reducing TNF-alpha levels and nuclear factor-kappaB activation.     

Stimulation of human peripheral blood mononuclear cells by the sialic acid precursor N-propanoylmannosamine

N-Propanoylmannosamine is an unnatural precursor of sialic acid, which is taken up by a variety of animal cells and metabolized to N-propanoylneuraminic acid. In several studies it has been demonstrated that application of unnatural precursors of sialic acids such as N-propanoylmannosamine (ManNProp) and homologues interfere with cell differentiation and proliferation of neuronal cells or embryonic stem cells. Since the function of the immune system is known to rely on the presence of sialic acid, we applied ManNProp to human peripheral blood mononuclear cells (PBMC). When culturing those lymphocytes with ManNProp 10 % of the natural sialic acid N-acetylneuraminic acid could be replaced by the newly formed N-propanoylneuraminic acid. This procedure resulted (a) in a marked stimulation in the rate of proliferation of PBMC, (b) a 10-fold increase of IL-2 production coupled with an up-regulation of its receptor CD25 on the cell surface and (c) a concomitant expression and regulation of the transferrin receptor with cell growth. The stimulation of PBMC by ManNProp might therefore introduce a new approach of immunomodulation.     

Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1 alpha

Human endothelial cells synthesize large amounts of platelet-activating factor (PAF) after 30-min treatment with recombinant tumor necrosis factor (TNF). Synthesis of PAF peaks at 4-6 h, whereas in endothelial cells treated with interleukin 1 alpha (IL-1) it peaks at 8-12 h. More than twice as much PAF is synthesized in response to optimal concentrations of TNF than in response to IL-1. However, PAF synthesis is stimulated by lower molar concentrations of IL-1 than TNF. About 30% of PAF produced in response to either TNF or IL-1 is released into the medium, whereas approximately 70% remains cell-associated. Experiments with labeled precursors show that PAF is synthesized de novo in response to TNF. This activity of TNF is inhibited by treating endothelial cells with the inhibitors of protein or RNA synthesis cycloheximide or actinomycin D. This finding may be explained by the observation that TNF induces in endothelial cells an acetyltransferase required for PAF synthesis. The induction of this enzymatic activity precedes the peak of PAF synthesis in TNF-treated cells. After prolonged incubation with either TNF or IL-1, endothelial cells no longer respond to the same monokine, but are still capable of producing PAF when treated with the other monokine. The finding that these monokines do not show reciprocal tachyphylaxis in endothelial cells may be explained by their binding to different receptors. In cells treated simultaneously with different concentrations of TNF and IL-1, PAF synthesis is stimulated in an additive rather than synergistic way. This suggests that PAF is synthesized by the same pathway in response to TNF or IL-1.     

Effects of an anti-tumor polysaccharide, schizophyllan, on interferon-gamma and interleukin 2 production by peripheral blood mononuclear cells

We examined the effect of schizophyllan, a neutral glucan isolated from the culture filtrate of Schizophyllum commune Fries, on the production of interferon-gamma (IFN-gamma) and interleukin 2 (IL 2) from the mitogen-stimulated human peripheral blood mononuclear cells (PBMC). When the levels of IFN-gamma and IL 2 in the culture medium of phytohemagglutin (PHA)- or concanavalin A (Con A)-stimulated PBMC were measured by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) respectively, a significant increase in the production of both cytokines by schizophyllan was demonstrated. These results suggest that the increased production of IFN-gamma and IL 2 may be responsible for the anti-tumor activity of this glucan.