The sensitizers nickel sulfate and 2,4-dinitrofluorobenzene increase CD40 and IL-12 receptor expression in a fetal skin dendritic cell line.

Dendritic cells (DCs) are antigen-presenting cells (APCs) capable of capturing haptens and to process and present them to T lymphocytes. In order to sensitize T cells for contact hypersensitivity (CHS), skin DCs suffer a maturation process with modifications on their surface molecules. The aim of this work was to evaluate changes induced by two contact sensitizers, 2,4-dinitrofluorobenzene (DNFB) and nickel sulfate (NiSO4), and a non-sensitizer 2,4-dichloronitrobenzene (DCNB), on the protein levels of two activation markers, CD40 and IL-12 receptor (IL-12R), in a mouse skin dendritic cell line (FSDC). The expression of CD40 and IL-12R proteins was evaluated by western blot assay and direct immunofluorescence microscopy. The results showed that CD40 and IL-12R expression increased significantly after cell exposure to NiSO4 and DNFB, although DNFB exhibited a stronger activity. There was no effect with DCNB. The epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), also used in the experiments, slightly increased the expression of both CD40 and IL-12R and when tested together with the sensitizers the effect was partially additive. The results suggest that the sensitizers DNFB and NiSO4 are directly involved on the changes of the surface markers CD40 and IL-12R in skin DCs, during the sensitization phase of CHS, and this effect may be enhanced by GM-CSF. In contrast, no effect was observed with DCNB.

Necroptosis is associated with low procaspase-8 and active RIPK1 and -3 in human glioma cells.

Necroptosis is a regulated necrotic cell death that involves receptor-interacting protein kinases RIPK1 and RIPK3. Here, we report that edelfosine triggers a rapid and massive cell death in human glioblastoma cells with characteristics of necrosis. Only a minor proportion of edelfosine-treated cells underwent caspase-dependent apoptosis. Autophagy and a rapid influx of extracellular calcium into the cells had little impact on cell death. Levels of procaspase-8 were very low in necroptosis-prone glioma cells compared with the levels in other cancer cell types that underwent apoptosis upon edelfosine treatment. The RIPK1-dependent necroptosis inhibitors necrostatin-1 (Nec-1) and Nec-1s as well as siRNA-mediated silencing of RIPK3 inhibited edelfosine-induced necroptosis, resulting in increased caspase-dependent apoptosis in edelfosine-treated glioblastoma U118 cells. Inhibition of the RIPK3 substrate MLKL with necrosulfonamide also increased apoptosis in edelfosine-treated cells. These data support a major role for RIPK1 and RIPK3 in the induction of necrotic cell death and in the switch from necrosis to apoptosis following edelfosine treatment. These results indicate that the ether lipid edelfosine exerts a rapid necroptotic cell death in apoptosis-reluctant glioblastoma cells, suggesting that induction of necroptosis could constitute a new approach for glioblastoma therapy.

Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: contribution of chlorogenic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon citratus (DC.) Stapf leaves infusion is used in traditional medicine for the treatment of inflammatory conditions, however little is known about their bioactive compounds. AIM OF THE STUDY: Investigate the compounds responsible for anti-inflammatory potential of Cymbopogon citratus (Cy) on cytokines production induced by lipopolysaccharide (LPS) in human and mouse macrophages, and the action mechanisms involved. MATERIALS AND METHODS: An essential oil-free infusion of Cy was prepared and polyphenol-rich fractions (PFs) were obtained from it by column chromatography. Chlorogenic acid (CGA) was identified, by HPLC/PDA/ESI-MS(n). The expression of cytokines, namely TNF-α and CCL5, was analyzed by real-time RT-PCR, on LPS-stimulated human macrophages. Activation of nuclear factor (NF)-κB, a master regulator of inflammation, was investigated by western blot and gene reporter assay. Proteasome activity was assessed using a fluorogenic peptide. RESULTS: Cymbopogon citratus extract and its polyphenols inhibited the cytokine production on human macrophages. This supports the anti-inflammatory activity of Cy polyphenols in physiologically relevant cells. Concerning the effect on the activation of NF-κB pathway, the results pointed to an inhibition of LPS-induced NF-κB activation by Cy and PFs. CGA was identified, by HPLC/PDA/ESI-MS(n), as the main phenolic acid of the Cy infusion, and it demonstrated to be, at least in part, responsible by that effect. Additionally, it was verified for the first time that Cy and PFs inhibited the proteasome activity, a complex that controls NF-κB activation, having CGA a strong contribution. CONCLUSIONS: The results evidenced, for the first time, the anti-inflammatory properties of Cymbopogon citratus through proteasome inhibition and, consequently NF-κB pathway and cytokine expression. Additionally, Cy polyphenols, in particular chlorogenic acid, were highlighted as bioactive compounds.