Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils

Background Priming of eosinophils. with granulocyte-macrophage colony-stimu lating factor (GM-CSF) and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a is associated with a rapid production of leukotrienes (LTs) and release of eosinophil cationic protein (ECP). Objective This study was designed to determine the effects of the sesquiter pene esters petasin, isopetasin and neopetasin on LT generation and ECP rel ease in eosinophils in vitro. Methods The model of eosinophil activation described above was used to indu ce LT production and ECP release. Cells were incubated with petasins and co ntrol inhibitors prior to priming and stimulation. To analyse intracellular steps of eosinophil activation and determine potential drug targets, some key signalling events were studied. Activity of cytosolic phospholipase A(2 ) (cPLA(2)) was measured by analysing the generation of arachidonic acid (A A). Translocation of 5-lipoxygenase (5-LO) was observed using immunofluores cence microscopy. Intracellular calcium concentrations [Ca2+](i) were measu red by a bulk spectrofluorometric assay. Results Whereas all three compounds inhibited LT synthesis, ECP release fro m eosinophils was blocked by petasin only, but not isopetasin or neopetasin . Similarly, PAF- or C5a-induced increases in [Ca2+](i) were completely abr ogated by petasin only, whereas isopetasin and neopetasin had significant l ower blocking efficacy. Moreover, only petasin, but not isopetasin or neope tasin, prevented increases in cPLA(2) activity and 5-LO translocation from the cytosolic compartment to the nucleus envelope in calcium ionophore-stim ulated eosinophils. Conclusion These data suggest that different petasins may at least partiall y block different intracellular signalling molecules. To reduce LT synthesi s, isopetasin and neopetasin may act at the level of or distal to 5-LO. In contrast, petasin may inhibit inflammatory effector functions in human eosi nophils by disrupting signalling events at the level of or proximal to phos pholipase C-beta (PLCbeta), besides its potential inhibitory activity withi n mitogen-activated protein kinase (MAPK) and LT pathways.