The p38 and ERK MAPK pathways are activated in human neutrophils by chemo attractants, pro-inflammatory cytokines, lipopolysaccharide (LPS), and Fcγ receptor ligation. Pharmacologic inhibition of p38 MAPK activation attenuates neutrophil respiratory burst activity, exocytosis, chemotaxis, adhesion, IL-8 synthesis and stress-induced apoptosis. Pharmacologic inhibition of ERK activity enhances neutrophil apoptosis, while the role of ERK in respiratory burst activity remains controversial [1]. Inhibition of both kinases is a promising therapeutic strategy to treat chronic inflammatory diseases.
The present study tested the hypothesis that eupatoriopicrin (sesquiterpene lactone, isolated from E.cannabinum L.) mediates multiple p38 and ERK1/2 MAPK-dependent responses in human neutrophils.
To verify the hypothesis we tested activity of eupatoriopicrin and positive controls (quercetin and clarithromycin) on LPS and f-MLP-stimulated neutrophils. We examined: (I) phosphorylation level of p38, ERK1/2 and JNK MAP kinases, (II) degranulation; respiratory burst activity and elastase release, (III) pro-inflammatory cytokine release (IL-8, IL-1β and TNFα) and (IV) apoptosis.
Phosphorylation of p38, ERK1/2 and JNK MAPK was determined by immunoblotting analysis. The inhibition of ROS production was determined using luminol dependent chemiluminescence method. Neutrophil elastase release was established spectrophotometrically. The effect on chemokines production was measured by enzyme-linked immunosorbent assay (ELISA). The apoptosis of neutrophils was analyzed with flow cytometry.
Inhibition of p38 kinase activity by eupatoriopicrin significantly attenuated degranulation and pro-inflammatory cytokine release. Respiratory burst and elastase activity were significantly inhibited by eupatoriopicrin at 2.5µM, compared with quercetin and clarithromycin tested at 50 µM . Lipopolysaccharide-induced IL-8 and TNFα production was significantly inhibited by eupatoriopicrin at 0.25 µM (IC50 < 1 µM). Inhibition of p38 and ERK1/2 activity by eupatoriopicrin at 0.25 µM resulted in an abolition of LPS-delay of neutrophils apoptosis.
These data suggest that eupatoriopicrin mediates both p38- and ERK MAPK-dependent neutrophil responses leading to potential beneficial health effects.
[1] Keshet Y et al. MAP Kinase Signaling Protocols 2010, 3-38