LPS-induced NO Release Research Articles

Contrasting effects of alendronate and clodronate on RAW 264 macrophages: the role of a bisphosphonate metabolite

Clodronate (dichloromethylidene-bisphosphonate), a halogen-containing bisphosphonate, can inhibit the release of cytokines from RAW 264 macrophages and has anti-inflammatory properties in rheumatoid arthritis, whilst amino-containing bisphosphonates such as alendronate (4-amino-1-hydroxybutylidene-bisphosphonate), have pro-inflammatory properties and can cause an acute phase response. The basis for these pharmacological properties is unclear. Recently, it was demonstrated that clodronate is metabolised by certain cell lines in vitro to an analogue of ATP, whereas amino-bisphosphonates are not. We therefore investigated whether clodronate can also be metabolised by RAW 264 macrophages and whether intracellular accumulation of the metabolite (AppCCl 2p) could account for the anti-inflammatory properties of clodronate. The effect of alendronate and AppCCl 2p on the release of cytokines (IL-1β, IL-6, and TNFα) from RAW 264 cells was compared, and the effect of the bisphosphonates and AppCCl 2p on the DNA binding activities of transcription factors, NF-κB and AP-1, was investigated. Pretreatment of RAW 264 macrophages with alendronate augmented the LPS-stimulated release of IL-1β and increased the binding of NF-κB to DNA in an electrophoretic mobility shift assay. Without LPS-induction, alendronate did not affect cytokine release or NF-κB binding. Clodronate was metabolised by RAW 264 cells to AppCCl 2p. Like clodronate, AppCCl 2p inhibited the LPS-induced release of cytokines and NO from RAW 264 macrophages. Both clodronate and its metabolite also inhibited the LPS-stimulated binding of NF-κB to DNA. In conclusion, these results suggest that the metabolite of clodronate may be responsible for the anti-inflammatory properties of clodronate, and that the contrasting effects of different bisphosphonates on the release of cytokines could be mediated partly through changes in the DNA binding activity of NF-κB.

Differential regulation of microglial activation by propentofylline via cAMP signaling

A pathological microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially toxic agents and by triggering reactive astrocytic changes. Using cultured microglia from neonatal rat brains, we investigated the mode of propentofylline action in strengthening cAMP-dependent intracellular signaling. We compared this action with the effects of dibutyryl-cAMP, a cell-permeable cAMP analog. Propentofylline inhibited lipopolysaccharide (LPS)-induced release of both tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a dose-dependent manner within the therapeutic low micromolar range. However, LPS-induced release of IL-6 and NO were not affected by propentofylline. All these differential effects of propentofylline on LPS-induced microglial release were mimicked by the addition of dibutyryl-cAMP. Microglial proliferation and phorbol myristate acetate (PMA)-induced O 2 − release were also dose-dependently inhibited by propentofylline as well as dibutyryl-cAMP. These results suggest that propentofylline, probably via reinforcement of cAMP intracellular signaling, alters the profile of the newly adopted immune properties in a way that it inhibits potentially neurotoxic functions while maintaining beneficial functions. This differential regulation of microglial activation may explain the neuroprotective mechanism exerted by propentofylline.

THE ROLE OF ENDOGENOUS IFN-γ, TNF-α AND IL-10 IN LPS-INDUCED NITRIC OXIDE RELEASE IN A MOUSE MODEL

Mice injected with lipopolysaccharide (LPS) develop lethal septic shock, accompanied by elevated serum NOx, interferon γ (IFN-γ), tumour necrosis factor α (TNF-α) and TNF-receptor levels. Elevated NO levels are thought to play a central role in tissue damage observed during septic shock. In vitro data indicate that IFN-γ and TNF-α play an important role in LPS-induced NO release. Further, interleukin 10 (IL-10) has been shown to inhibit the release of pro-inflammatory cytokines such as IFN-γ and TNF-α. Therefore, in the present study, we investigated the role of IFN-γ, TNF-α, and IL-10 in LPS-induced NO release. To this end, mice were pretreated with anti-IFN-γ, anti-TNF-α, anti-IL-10 mAbs or combinations of these 2 h before LPS-challenge. The results indicate that IFN-γ, TNF-α as well as IL-10 are involved in the regulation of LPS-induced NO release. Blocking either IFN-γ or TNF-α has no effect on LPS-induced NO release, however, blocking both IFN-γ and TNF-α nearly completely prevents NO release after LPS challenge, suggesting that the presence of either TNF-α or IFN-γ is essential for induction of NO release after LPS challenge. Further, the results obtained with anti-IL-10 treatment suggest the presence of an IL-10 inducible factor which together with IFN-γ and TNF-α regulates LPS-induced NO release.

The influence of age, work schedule and personality on morningness dimension

Two new compounds, named carpabrotalactone C (1) and methyl 8-[2(5H)-furanon-5-yl]octanoate (2), were isolated from the herbs of Carpesium abrotanoides L. The structures of these compounds were elucidated based on spectroscopic analysis, single crystal X-ray diffraction analysis and ECD calculation. Compound 1 exhibited potent cytotoxicity against the A549, SMMC-7721, MCF-7 and SW480 cell lines, with IC50 values from 11.46 to 23.68 μM. Meantime, compound 1 showed weak inhibitory eff ;ects on LPS-induced NO release in RAW 264.7 cell lines, with IC50 value of 63.23 ± 2.55 μM.