Characterisation of granulocyte stimulation by thionins from European mistletoe and from wheat

Thionins are small basic peptides found in different plant species, which a re known to exert cytotoxic properties. In addition, previous data indicate d an activation of human granulocytes by thionins from European mistletoe ( viscotoxins, VT). To extend these latter findings, we investigated the infl uence of VT and from thionins from wheat flour (purothionin) on human granu locytes by flow cytometry and tried to characterise the involved molecular structures and mechanisms. Phagocytosis was determined by incorporation of FITC-labelled Escherichia coli and respiratory burst by oxidation of dihydr orhodamine 123 to rhodamine 123. VT and purothionin significantly enhanced E. coil-stimulated phagocytosis and respiratory burst at 25 and 250 mu g/ml . Phagocytosis of damaged lymphocytes by granulocytes was detected by elect ron microscopy in the VT-stimulated (100 mu g/ml) but not in the control cu ltures. The poly-cationic structure of the intact ed by comparison of the b urst and phagocytosis-enhancing effects induced by other poly-cationic (pro tamine sulphate, histone, poly-L-arginine, poly-L-lysine) and poly-anionic (poly-L-glutamic acid) peptides, while pore forming due to amphipathic prop erties seems to be less important. Ca2+ and Mg2+ could not inhibit VT-enhan ced phagocytosis and, thus, could not inhibit binding of VT to granulocytes . In addition, verapamil at low concentrations inhibited VT activity, sugge sting the involvement of Ca2+ channels for granulocyte activation by the VT . Similarly, thionins and histones in contrast to protamine sulphate induce d cell death of granulocytes at 250 mu g/ml as demonstrated by an enhanced release of reactive oxygen intermediates in unstimulated granulocytes. From these data one may suggest that activity of VT is induced by strong unspec ific ionic binding, probably followed by specific receptor binding, and thi onins exhibit stimulatory and cytotoxic effects on immune cells, which have to be further characterised. (C) 1999 Elsevier Science B.V. All rights res erved.