Specific activation of human neutrophils by scorpion venom: A flow cytometry assessment

Acute lung injury following envenomation by Tityus scorpion species is due in part to activation of the inflammatory response leading to release of cytotoxic leukocyte-derived products, including cytokines and possibly reactive oxygen species (ROS). Tityus zulianus envenomation in Venezuela produces cardiorespiratory complications and death by lung injury whereas stings by Tityus discrepans produce mainly gastrointestinal and pancreatic alterations. To ascertain the role played by granulocytes in the envenomation by T. zulianus (TzV) and T. discrepans (TdV), human peripheral blood neutrophils, eosinophils, and monocytes were exposed to scorpion venoms (0.001-5 μg/mL) and the kinetics (5-15 min) of peroxide production determined by flow cytometry, using 2′,7′-dichlorodihydrofluorescein diacetate (succinimidyl ester) as a fluorescent substrate. TzV induced a significantly (p < 0.01) more potent increase in peroxide production in neutrophils (for 5 and 10 min of incubation), and to a lesser extent in monocytes (5-15 min), compared to TdV. TzV induced necrosis in neutrophils at doses higher than 5 μg/mL. No effect was observed on eosinophils, suggesting that TzV specifically targets neutrophil intracellular ROS production. The TzV-stimulated pathway is protein kinase C-dependent because it was almost completely (>90%) abolished by staurosporine. The stimulatory effect is associated with the lowest molecular mass venom peptides as gel filtration fractions TzII and TzIII significantly enhanced peroxide production. The combined used of the intracellular ROS agonist, phorbol myristate acetate (PMA), and TzV produced a modest but significant increase in peroxide production suggesting the possibility of overlapping signaling cascades amongst PMA and TzV. Up-regulation of intracellular neutrophil ROS production may be an important in vivo target for TzV which could have a role to play in the cardiorespiratory complications elicited after envenomation by this species.