Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages

Apigenin and its structural analogues chrysin and luteolin were used to evaluate their capacity to inhibit the production of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC). Furthermore, flowcytometric analysis was performed to compare the effects of apigenin, chrysin, luteolin, quercetin and naringenin on the different cell types present in PBMC. LPS-stimulated PBMC were cultured in the presence of the flavonoids and TNFalpha, IL-1beta and IL-6 were measured in the supernatants. In parallel, metabolic activity of the PBMC was determined by measuring succinate dehydrogenase activity. Apigenin, chrysin and luteolin dose-dependently inhibited both pro-inflammatory cytokine production and metabolic activity of LPS-stimulated PBMC. With increasing concentration of apigenin, chrysin or luteolin the monocytes/macrophages disappeared as measured by flowcytometry. This also appeared to occur in the non-LPS-stimulated PBMC. At the same time there was an increase in dead cells. T- and B-lymphocytes were not affected. Quercetin and naringenin had virtually no effects on cytokines, metabolic activity or on the number of cells in the studied cell populations. In conclusion, monocytes were specifically eliminated in PBMC by apigenin, chrysin or luteolin treatment in vitro at low concentrations (around 8 microM), in which apigenin appeared to be the most potent.     

Potencies of topical glucocorticoids to mediate genomic and nongenomic effects on human peripheral blood mononuclear cells

Several different genomic and nongenomic mechanisms are known to mediate the important anti-inflammatory and immunomodulatory effects of glucocorticoids (GC). Genomic effects are the most important while the clinical relevance of nongenomic actions is still a matter of debate. We therefore investigated whether beclometasone and clobetasol are particularly suitable for topical application because of their specific spectrum of genomic and nongenomic actions. For these purposes we compared effects on oxygen consumption as measured with a Clark electrode (nonspecific nongenomic glucocorticoid effects), on interleukin-6 synthesis by means of ELISA (genomic effects) and on apoptosis using flow cytometry (nongenomic and genomic effects) in quiescent and mitogen-stimulated PBMC. Beclometasone and clobetasol indeed had stronger effects on the oxygen consumption of quiescent and stimulated cells at lower concentrations (10(-10) and 10(-8) M) but were less potent at higher concentrations (10(-5) and 10(-4) M) in comparison with dexamethasone. Also in terms of genomic potency, topical GC were more effective than dexamethasone at 10(-10) and 10(-8) M but gave similar results at higher concentrations. The ability of all three GC to induce apoptosis was found to be concentration-dependent and similar at concentrations between 10(-8) and 10(-5) M. But, compared with 10(-4) M dexamethasone, topical GC at 10(-4) M were significantly more effective at inducing apoptosis in both PBMC and Jurkat T-cells. These results show that topical GC have different concentration--(genomic/nongenomic) effect--ratios compared with dexamethasone: besides to the well-known genomic effects there are also significant nongenomic effects of topical glucocorticoids that already at low concentrations might be more therapeutically relevant in certain clinical conditions than currently assumed.     

Effects of fumonisin B(1) on the expression of cytokines and chemokines in human dendritic cells.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other crops across the world. Exposure to FB(1) is known to have toxic and carcinogenic effects in animals, and to express toxicity in cells. In this study, we investigated mechanisms whereby FB(1) may induce immunotoxic effects in human dendritic cells (DC) differentiated from human peripheral blood mononuclear cells. mRNA and protein levels of a number of cytokines and chemokines were analyzed in DC, after exposure to 100 microM FB(1), 10 ng/ml LPS, or a combination of 100 microM FB(1) and 10 ng/ml LPS for 6h or 24h. Exposure to FB(1) resulted in an increase in the expression of IFNgamma and CXCL9. Moreover, FB(1) inhibited the LPS-induced expression of IL-6, IL-1beta, CCL3 and CCL5. The other cytokines studied (TNFalpha, IL-12, IL-18 and IL-23) were not affected by FB(1) in DC. The results of this study indicate that FB(1) has an impact on the expression of cytokines and chemokines in human DC, and in addition to its other toxic effects, FB(1) may also be potentially immunotoxic to humans.     

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte-derived dendritic cells

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides - which are the most frequent cause of adverse drug reactions - were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines.     

Low levels of the air pollutant 1-nitropyrene induce DNA damage,increased levels of reactive oxygen species and endoplasmic reticulum stress in human endothelial cells

Both epidemiological and experimental studies suggest that exposure to high levels of air pollution is a risk factor associated with cardiovascular disease. Traffic emission is a major source of exposure to persistent air pollutants such as nitrated polycyclic aromatic hydrocarbons (nitro-PAHs). 1-Nitropyrene (1-NP), one of the most abundant nitro-PAHs in diesel exhausts, was selected as a model nitro-PAH for the present study. The aim of the study was to investigate the effects of 1-NP in human umbilical vein endothelial cells (HUVECs) and the metabolic pathways involved. The nitroreductase inhibitor dicoumarol and the coplanar aryl hydrocarbon receptor (AhR) ligand PCB 126 were used to modulate the metabolism of 1-NP. The results revealed that low levels (≤10 μM) of 1-NP induced DNA damage, increased levels of reactive oxygen species (ROS) and increased protein expression of the endoplasmic reticulum (ER) stress chaperone GRP78. A decrease in cell viability was only observed following exposure to a higher level of 1-NP (15 μM). Inhibition of nitroreductive metabolism by dicoumarol attenuated the induction of DNA damage, intracellular ROS levels and GRP78 expression. This suggests that the effects of 1-NP on HUVEC were mediated by metabolites mainly formed at nitroreduction. Our findings suggest that the human blood vessel endothelium is a sensitive target tissue for the major nitro-PAH constituent in diesel exhaust.     

Effect of sesquiterpene lactones on the expression of the activation marker CD69 and of IL-2 in T-lymphocytes in whole blood

We used flow cytometry to investigate the inhibitory effect of sesquiterpene lactones (SLs) on T-cell activation measured by the expression of its early marker CD69, and on interleukin (IL)-2, a mediator of activation, in whole blood. SLs are biologically active compounds found especially in plants from the Asteraceae family. Overnight treatment of blood with these substances led to the inhibition of CD69 and IL-2 expression. Interestingly, bifunctional SLs showed a weaker activity than monofunctional substances, which is in contradiction with the data obtained so far, using other biological test systems. Additionally, SLs did not completely inhibit CD69 or IL-2 expression. We also determined their toxicity and observed only a low effect. Up to now, studies on cytotoxicity have only been performed using cultured cell lines. From these results it may be supposed that these natural compounds preferentially show toxic effects towards transformed cell lines. Altogether, the results demonstrated that SLs effectively inhibit the activation of the T-lymphocyte response in whole blood and proved the utility of a whole blood system in studying their biological effects.