Cyclic nucleotides differentially regulate the synthesis of tumour necrosis factor-alpha and interleukin-1 beta by human mononuclear cells.

Recent reports have shown that phosphodiesterase (PDE) inhibitors suppress production of tumour necrosis factor-alpha (TNF-alpha) in mouse macrophages. In the present study we show that theophylline, pentoxifylline and 3-isobutyl-1-methylxanthine markedly suppress the lipopolysaccharide (LPS)-induced synthesis of TNF-alpha (also) in human mononuclear cells. This effect is selective for TNF-alpha since up to several-fold higher concentrations of these PDE inhibitors do not affect production of interleukin-1 beta (IL-1 beta) in the same system. The observed effect of PDE inhibitors appears to be mediated by accumulation of cAMP since (i) addition of PDE inhibitors increases cAMP while cGMP levels are only marginally elevated; (ii) raising cAMP by another mechanism (enhanced formation induced by prostaglandin E2; PGE2) leads to a similar suppression of TNF-alpha production; and (iii) raising cGMP by activating the soluble guanylate cyclase by 3-morpholinosydnonimine (SIN 1) does not inhibit TNF-alpha synthesis. However, SIN 1 suppressed the synthesis of IL-1 beta. Selective suppression of TNF-alpha synthesis by PDE inhibitors may contribute to their beneficial effects in animal models of septic shock or lung injury and may thus have clinical implications.     

Enhanced tumor necrosis factor suppression and cyclic adenosine monophosphate accumulation by combination of phosphodiesterase inhibitors and prostanoids

We investigated cooperative effects of phosphodiesterase (PDE) inhibitors and prostanoids on cyclic adenosine monophosphate (cAMP) accumulation and tumor necrosis factor (TNF)-α synthesis in human peripheral blood mononuclear cells (PBMC). PDE inhibitors alone induced only a small increase in cAMP levels in lipopolysaccharide (LPS)-stimulated PBMC. Cicaprost (a stable analogue of prostacyclin) and pentoxifylline added simultaneously to LPS-stimulated PBMC (2.0 × 10⁶/ml) induced a rapid increase of cAMP to a level of 100 nM that peaked within 10 min and remained at a plateau for up to 4 h. Thus combined prostanoids and PDE inhibitors enhanced cAMP accumulation. TNF-α suppression in the presence of pentoxifylline and prostanoids exceeded that of either drug alone. The potency of different PDE inhibitors (theophylline, pentoxifylline, penthy-droxifylline, albifylline, torbafylline, A 802715, amrinone and rolipram) to increase cAMP levels in combination with cicaprost was evaluated after 1 h of incubation. The dose-dependent increase of cAMP for all PDE inhibitors tested in this combined stimulation provided a useful tool for evaluating the potency of PDE inhibitors on cAMP accumulation. The effective concentration of PDE inhibitors, which raised cAMP levels to 300% of control, (EC³⁰⁰), correlated with the IC₅₀ for TNF-α suppression (r = 0.930, p = 0.007, with theophylline excluded from the analysis). Interestingly, by contrast, the specific type IV PDE inhibitor rolipram caused only a moderate rise of accumulated cAMP in the same cells. Our data support cAMP as an essential mediator for TNF-α suppression by PDE inhibitors. Furthermore, an enhanced inhibiting effect on TNF-α production may prove therapeutically advantageous. It may occur in inflammatory and infectious diseases in vivo, since high levels of endogenous prostaglandins are liberated in these conditions.     

Anti-inflammatory activity of salmeterol: down-regulation of cytokine production.

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.     

Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.     

Effects of thiazinamium chloride on histamine release from rat peritoneal mast cells and on phosphodiesterase activity in guinea pig lung

The effect of thiazinamium Cl (TCl) on histamine release from rat peritoneal mast cells (RPMC) was investigated. Although TCl inhibited compound 48/80-induced histamine release moderately (IC50 value 40 microM), the drug was a weaker inhibitor of ovalbumin-induced histamine release (100 microM, -21%). In contrast, promethazine HCl (PHCl) was more effective against antigen-induced histamine release (IC50 value 13 microM) than against compound 48/80-induced histamine release (100 microM, -53%). Disodium cromoglycate (DSCG) was effective against both antigen and compound 48/80-induced release of histamine with IC50 values of 7 and 1 microM, respectively. Neither TCl nor DSCG at 1 mM increased spontaneous release of histamine from RPMC, whereas PHCl induced spontaneous release by over 50% at 1 mM. TCl did not inhibit phosphodiesterase (PDE) activity in guinea pig lung at 1 mM, whereas theophylline and DSCG inhibited PDE with IC50 values of 1.1 and 0.32 mM, respectively. These data suggest that high local concentrations of TCl may reduce histamine release during an asthmatic attack and improve its effectiveness as a bronchoprotectant.     

Anti-allergic effects of milrinone and sulmazole in isolated rat lungs in comparison with theophylline

Milrinone and sulmazole, two recently developed drugs, inhibit specific fractions of the phosphodiesterase (PDE) isozyme system. Since theophylline aspecifically inhibits the PDE complex, we compared the effects of milrinone and sulmazole with those of theophylline on antigen-induced bronchoconstriction, vasoconstriction, mediator release and leukotriene production. In the isolated perfused and ventilated lung of actively sensitized rats, we elicited antigen-induced bronchoconstriction, vasoconstriction and release of mediators like histamine, 5-hydroxytryptamine (5-HT) and slow-reacting substance of anaphylaxis (SRS-A). Milrinone, sulmazole and theophylline inhibited antigen-induced bronchoconstriction and vasoconstriction in a dose-dependent manner with minor differences in potency. Antigen-induced release of preformed mediators like histamine and 5-HT was inhibited only at high concentrations of milrinone, whereas sulmazole failed to inhibit mediator release. Theophylline also failed to inhibit 5-HT release. However, SRS-A synthesis was markedly reduced by these drugs in relatively low concentrations. It is concluded that milrinone and sulmazole have anti-allergic effects similar to those of theophylline and that all three PDE inhibitors reduce SRS-A synthesis.     

Design, synthesis, and preliminary evaluation of new pyrrolidine derivatives as neuraminidase inhibitors

A series of pyrrolidine derivatives were designed and synthesized in good yields starting from commercially available 4-hydroxy-L-proline using a suitable synthetic strategy. And their ability to inhibit neuraminidase was evaluated. These compounds showed potent inhibitory activity against influenza A (H3N2) neuraminidase. Within this series, four compounds, 6e, 9c, 9f and 10e, have the good potency (IC(50)=1.56 approximately 2.40microM) which is compared to the NA inhibitor oseltamivir (IC(50)=1.06microM), and could be used as lead compound in the future.     

Inhibition of endotoxin-induced TNF-alpha production in macrophages by 5Z-7-oxo-zeaenol and other fungal resorcylic acid lactones

Resorcylic acid lactones are fungal metabolites that exhibit a wide range of biological properties which includes oestrogenic, antifungal, phytotoxic and anti-inflammatory activity. The capacity of 5Z-7-oxo-zeaenol, a resorcylic lactone of fungal origin and six naturally occurring analogues to inhibit lipopolysaccharide (LPS)-induced cytokine production in phorbol 12-myristate-13-acetate (PMA)-treated cultured myelomonocytic cells (U937) was compared. The activity of the natural analogues in the U937 assay varied over 10(4)-fold, with 5Z-7-oxo-zeaenol the most potent of those tested inhibiting tumour necrosis factor-alpha (TNF alpha) production in these cells with IC50 of 6 nM. The isomeric 7-oxo-zeaenol and structurally more distant monorden (radicicol) were the next most active compounds with IC50 approximately 500 nM, and zearalenone, the least active with IC50 > 400 microM. 5Z-7-oxo-zeaenol retained activity in LPS-stimulated peripheral blood mononuclear cells with an IC50 of 10-25 nM. This compound also inhibited LPS-induced TNF alpha production in whole blood experiments (IC50 100-1000 nM) and lowered serum levels of TNF alpha in mice when administered prior to LPS. 5Z-7-oxo-zeaenol was shown to inhibit the phosphorylation and activation of mitogen-activated protein kinase (MAPK) induced by LPS. These data are consistent with a mechanism of action at or upstream of MAPK with resultant downstream effects. This series of naturally occurring analogues represents an interesting group of compounds with diverse biological properties. Of this series, 5Z-7-oxo-zeanenol has exceptionally potent anti-inflammatory properties exhibited by its strong inhibition of cytokine production.     

Effect of lisofylline and pentoxifylline on the bacterial-stimulated production of TNF-alpha, IL-1 beta IL-10 by human leucocytes.

The present study concerns the effect of the xanthine derivates lisofylline (LSF) and pentoxifylline (PTX) on the production of pro-inflammatory cytokines tumour-necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) and the de-activating cytokine interleukin-10 (IL-10) by human leucocytes during stimulation with lipopolysaccharide (LPS), heat-killed Gram-negative bacteria (GNB) or Gram-positive bacteria (GPB). The production of TNF-alpha and IL-1 beta by leucocytes stimulated with LPS, Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae was inhibited by both drugs. The production of IL-10 by leucocytes stimulated with LPS and Hib was inhibited by both xanthine derivates only at 48 hr. However, incubation of leucocytes with S. pneumoniae in the presence of LSF or PTX stimulated the production of IL-10 about four- and twofold at 24 hr and 48 hr, respectively. In all instances, the extent of inhibition or enhancement of cytokine production by LSF or PTX was equal. The divergent effects of xanthine derivates on the IL-10 production indicate the existence of distinct intracellular pathways depending on whether leucocytes are stimulated by GPB or GNB.     

Bromelain inhibits lipopolysaccharide-induced cytokine production in human THP-1 monocytes via the removal of CD14

Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. However, the anti-inflammatory mechanism of bromelain is unclear. Therefore, we investigated the effect of bromelain on cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) and monocytic leukemia THP-1 cells. The result showed that bromelain (50-100 microg/ml) significantly and reversibly reduced tumor necrosis factor (TNF)-alpha interleukin- (IL)-1beta and IL-6 from LPS-induced PBMC and THP-1 cells. This effect was correlated with reduced LPS-induced TNF-alpha mRNA and NF-kappaB activity in THP-1 cells. In addition, bromelain dose-dependently inhibited LPS-induced prostaglandin E(2), thromboxane B(2) and COX-2 mRNA but not COX-1 mRNA. Importantly, bromelain degraded TNF-alpha and IL-1beta molecules, reduced the expression of surface marker CD14 but not Toll-like receptor 4 from THP-1 cells. Taken together, the results suggest that the suppression of signaling pathways by bromelain's proteolytic activity may contribute to the anti-inflammatory activity of bromelain.     

Cytokine inhibition by a novel steroid, mometasone furoate

Mometasone furoate (9 alpha, 21 dichloro-11 beta, 17 alpha dihydroxy-16 alpha methyl-1,4 pregnadiene-3, 20 dione-17-[2'] furoate) was an unexpectedly potent inhibitor of the in vitro production of three inflammatory cytokines, IL-1(1), IL-6, and TNF-alpha. The potency of mometasone furoate in inhibiting cytokine production was compared to that of hydrocortisone, betamethasone, dexamethasone, and beclomethasone. IL-6 and TNF-alpha were both produced by WEHI-265.1 (murine myelomonocytic leukemia) cells following stimulation by lipopolysaccharide (LPS). Twenty-four hours after stimulation by LPS, the cell-free supernatant fluids were removed. Their cytokine content was analyzed using ELISAs specific for each cytokine. IL-1 synthesis was induced in the harvested peritoneal macrophages of BALB/c mice by incubation with LPS for twenty-four hours. The IL-1 content in the cell-free supernatant fluids was determined by the thymocyte-costimulator bioassay. Using these systems, mometasone furoate was found to be the most potent steroid tested for inhibiting the production of the three cytokines. The IC50's were 0.05 nM (IL-1), 0.15 nM (IL-6), and 0.25 nM (TNF-alpha). The inhibition of the production of proinflammatory mediators by extremely low concentrations of mometasone furoate suggests that this steroid should be highly effective in various disorders.     

The specific type IV phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-α production by human mononuclear cells

Compounds suppressing the production of tumor necrosis factor-α are protective in animal modelsof septic shock. Recent studies demonstrated a beneficial effect of xanthine derivatives, which suppress tumor necrosis factor-α production by acting as non-specific cAMP phosphodiesterase inhibitors. In this experiment wwe tested the effect (±)-rolipram (racemate) and its enantiomers on human mononuclear cells stimulated with lipopolysaccharide (LPS). Rolipram has a phenyl-pyrrolidinone structure, unrelated to the methylxanthines, and acts as a specific inhibitor of the type IV phosphodiesterase. Our results identify rolipram as a remarkably potent suppressor of the LPS-induced synthesis of tumor necrosis factor-α. When compared to the non-specific inhibitor pentoxifylline, the IC₅₀ of (±)-rolipram (130 nM) is more than 500 times lower. The influence of rolipra on tumor necdrosis factor-α production depended on the steric configuration of the molecule, since the (−)-enantiomer exhibited a five times lower IC₅₀ than the (+)-enantiomer. The inhibitory effect of all substances tested is selective for tumor necrosis factor-α rather than interleukin-1β, since interleukin-1β production is only slightly influenced.     

Effects of isoflavones and soybeans fermented with Bacillus subtilis on lipopolysaccharide-induced production of tumor necrosis factor-alpha and fibrinolysis in vivo

The effects of isoflavones and of a derivative of soybeans fermented with Bacillus subtilis, designated Nattoesse, on the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha) and fibrinolysis were investigated in vivo. The dietary supplement Nattoesse contains several isoflavones. Therefore, we examined the effects of individual isoflavones (daidzein, daidzin, genistein, and genistin) on the LPS-induced production of TNF-alpha. Intraperitoneal injections of daidzein, daidzin, and genistin (but not of genistein before a challenge with LPS) resulted in significant depression of serum levels of TNF-alpha in mice. Daidzein had the strongest activity in this assay. Oral administration of daidzein to mice also had a significant suppressive effect, as compared with that of the Citrus flavanone naringin. In galactosamine-sensitized mice, by contrast, the suppression of LPS-induced lethal shock by daidzein was very weak. Nattoesse did not inhibit the production of TNF-alpha nor did it prevent lethal shock. However, oral administration of Nattoesse to mice significantly suppressed LPS-induced increases in scores of the fibrin degradation product, and the effect was both dose- and time-dependent. Thus, it appears that Nattoesse has fibrinolytic activity during LPS-induced circulatory failure.     

Biochemical study on anti-inflammatory action of anti-allergic drugs--with regard to NADPH oxidase]

The effects of anti-allergic drugs with or without H1-receptor antagonism on the NADPH oxidase from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of three drugs required for 50% inhibition of the oxidase (IC50) were as follows: azelastin--0.7 microM in the whole-cell system and 0.5 microM in the cell-free system, ketotifen--60 microM in the whole-cell system and 6.8 microM in the cell-free system, and oxatomide--25 microM in the whole-cell system and 9.7 microM in the cell-free system. In addition, in the cell-free system, these drugs did not change the Km values for the NADPH of the oxidase. However, micromoles of tranilast, an anti-allergic drug without H1-receptor antagonism, did not inhibit neutrophil NADPH oxidase in the whole-cell and cell-free systems. The IC50 of hydrocortisone in the cell-free system was 60 microM. These results suggest that anti-allergic drugs with H1-receptor antagonism inhibit reconstitution of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have a strong anti-inflammatory action. Anti-allergic drugs are not the drugs of first choice for asthma, but these drugs, especially basic anti-allergic drugs, should be used more frequently for the treatment of chronic asthma, infectious-typed asthma and mixed-typed asthma closely associated with acute and chronic inflammation of the airways as well as atopic asthma.     

Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

Involvement of the membrane form of tumour necrosis factor-alpha in lipopolysaccharide-induced priming of mouse peritoneal macrophages for enhanced nitric oxide response to lipopolysaccharide.

We studied the pathways of macrophage response to lipopolysaccharide (LPS). When mouse macrophages pre-exposed to LPS were restimulated with this agent, reduced tumour necrosis factor-alpha (TNF-alpha) responses (desensitization/endotoxin tolerance) were accompanied by increased (priming) nitric oxide (NO) responses. Priming was also inducible with recombinant interferon-beta (IFN-beta). The requirement of TNF-alpha biosynthesis in the LPS-induced priming was also suggested by the observation that both anti-TNF-alpha serum and pentoxifylline inhibited this effect. However, addition of mouse recombinant TNF-alpha (mrTNF-alpha) did not enhance the priming induced by LPS or IFN-beta, and preincubation with mrTNF-alpha alone, or in association with other cytokines produced by macrophages (interleukin-1 beta, interleukin-6, or leukaemia inhibitory factor), did not induce a priming effect. We found however, that pentoxifylline, which blocked the priming, also decreased the level of membrane-bound TNF-alpha. Furthermore, exposure to compound BB-3103 (a metalloproteinase inhibitor that blocks the processing of membrane-bound TNF-alpha yielding to the secreted cytokine) enhanced the priming effect, the expression of membrane TNF-alpha and the specific binding of LPS. These observations suggest that the membrane form of TNF-alpha is involved in the interaction of LPS with a receptor required for LPS-induced priming.