Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils.
BACKGROUND
Priming of eosinophils with granulocyte-macrophage colony-stimulating 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 sesquiterpene esters petasin, isopetasin and neopetasin on LT generation and ECP release in eosinophils in vitro.


METHODS
The model of eosinophil activation described above was used to induce LT production and ECP release. Cells were incubated with petasins and control 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 A2 (cPLA(2)) was measured by analysing the generation of arachidonic acid (AA). Translocation of 5-lipoxygenase (5-LO) was observed using immunofluorescence microscopy. Intracellular calcium concentrations [Ca2+]i were measured by a bulk spectrofluorometric assay.


RESULTS
Whereas all three compounds inhibited LT synthesis, ECP release from eosinophils was blocked by petasin only, but not isopetasin or neopetasin. Similarly, PAF- or C5a-induced increases in [Ca2+]i were completely abrogated by petasin only, whereas isopetasin and neopetasin had significant lower blocking efficacy. Moreover, only petasin, but not isopetasin or neopetasin, prevented increases in cPLA(2) activity and 5-LO translocation from the cytosolic compartment to the nucleus envelope in calcium ionophore-stimulated eosinophils.


CONCLUSIONS
These data suggest that different petasins may at least partially block different intracellular signalling molecules. To reduce LT synthesis, isopetasin and neopetasin may act at the level of or distal to 5-LO. In contrast, petasin may inhibit inflammatory effector functions in human eosinophils by disrupting signalling events at the level of or proximal to phospholipase Cbeta (PLCbeta), besides its potential inhibitory activity within mitogen-activated protein kinase (MAPK) and LT pathways.
Thomet
O A
OA
Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland.
Wiesmann
U N
UN
Blaser
K
K
Simon
H U
HU
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001798:Blood Proteins / Q000037:antagonists & inhibitors; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D015936:Complement C5a / Q000037:antagonists & inhibitors; D003545:Cysteine / Q000037:antagonists & inhibitors; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000517:prevention & control; D020024:Leukotriene Antagonists; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors; D012260:Ribonucleases; D012717:Sesquiterpenes / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D001798:Blood Proteins; D004791:Enzyme Inhibitors; D047091:Eosinophil Granule Proteins; D020024:Leukotriene Antagonists; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D010972:Platelet Activating Factor; D012717:Sesquiterpenes; C112381:cysteinyl-leukotriene; D015936:Complement C5a; C418727:petasin; C418728:isopetasin; D012260:Ribonucleases; D010741:Phospholipases A; D054467:Phospholipases A2; D003545:Cysteine; D002118:Calcium